Anti-transferrin receptor (TFR) antibody and uses thereof

ABSTRACT

Aspects of the disclosure relate to antibodies that bind to transferrin receptor (e.g., transferrin receptor 1) and complexes comprising the antibody covalently linked to a molecular payload. Methods of using the antibodies are also provided.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 63/220,043, filed Jul. 9, 2021,entitled “ANTI-TRANSFERRIN RECEPTOR (TFR) ANTIBODY AND USES THEREOF,”which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present application relates to novel anti-transferrin receptor (TfR)antibodies and the use of the antibodies.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(D082470076US02-SEQ-ZJG.xml; Size: 135,875 bytes; and Date of Creation:Jul. 1, 2022) is herein incorporated by reference in its entirety.

BACKGROUND

Transferrin Receptor (TfR) is a dimeric transmembrane glycoproteinreceptor involved in iron transport. Two transferrin receptors have beencharacterized in humans, transferrin receptor 1 (TfR1) and transferrinreceptor 2 (TfR2). It has been shown that TfR is overexpressed in cancercells with higher metastatic potential. TfR1 has been shown to expresson the endothelial cells of the blood brain barrier can be used to allowthe delivery of large molecules into the brain.

SUMMARY

The present disclosure is based, at least in part, on the development ofhumanized antibodies that bind transferrin receptor (anti-TfRantibodies). In some embodiments, anti-TfR antibodies described hereinselectively bind to human or non-human primate (NHP) transferrinreceptor 1 (TfR1) with high specificity and affinity (e.g., subnanomolarto nanomolar range). In some embodiments, the anti-TfR antibodiesdescribed herein are useful for targeting tissues and/or (e.g., and)cells that express TfR1. In some embodiments, the anti-TfR antibodiesprovided herein are used for detection of TfR1 in a cell or a tissue. Insome embodiments, the anti-TfR antibodies provided herein are used indiagnostic, therapeutic, or research applications. In some embodiments,the anti-TfR antibodies described herein are used to deliver a molecularpayload to a target cell or tissue (e.g., a cell or tissue thatexpresses TfR1).

As such, in some aspects, complexes comprising the anti-TfR antibodiesconjugated (e.g., covalently conjugated) to a molecular payload (e.g., adiagnostic agent or a therapeutic agent) are provided. In someembodiments, the anti-TfR antibodies is used to deliver the conjugatedmolecular payload to a cell or a tissue that expresses TfR1 (e.g.,muscle or the brain) for diagnosing and/or (e.g., and) treating adisease (e.g., a muscle disease or a neurological disease). In someaspects, the present disclosure provides data demonstrating that theanti-TfR antibodies described herein has superior activity in deliveringmolecular payload into a target cell (e.g., a muscle cell), comparedwith other known anti-TfR antibodies.

One aspect of the present disclosure relates to an antibody that bindsto human transferrin receptor (TfR), wherein the antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 76; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 75;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 69; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 70;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 74;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 75;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 74;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 95% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 95%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 95% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 95% identical toSEQ ID NO: 80.

In some embodiments, the antibody comprises:

(i) a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VLcomprising the amino acid sequence of SEQ ID NO: 75;

(ii) a VH comprising an amino acid sequence of SEQ ID NO: 69 and a VLcomprising an amino acid sequence of SEQ ID NO: 70;

(iii) a VH comprising an amino acid sequence of SEQ ID NO: 71 and a VLcomprising an amino acid sequence of SEQ ID NO: 70;

(iv) a VH comprising an amino acid sequence of SEQ ID NO: 72 and a VLcomprising an amino acid sequence of SEQ ID NO: 70;

(v) a VH comprising an amino acid sequence of SEQ ID NO: 73 and a VLcomprising an amino acid sequence of SEQ ID NO: 74;

(vi) a VH comprising an amino acid sequence of SEQ ID NO: 73 and a VLcomprising an amino acid sequence of SEQ ID NO: 75;

(vii) a VH comprising an amino acid sequence of SEQ ID NO: 76 and a VLcomprising an amino acid sequence of SEQ ID NO: 74;

(viii) a VH comprising an amino acid sequence of SEQ ID NO: 77 and a VLcomprising an amino acid sequence of SEQ ID NO: 78;

(ix) a VH comprising an amino acid sequence of SEQ ID NO: 79 and a VLcomprising an amino acid sequence of SEQ ID NO: 80; or

(x) a VH comprising an amino acid sequence of SEQ ID NO: 77 and a VLcomprising an amino acid sequence of SEQ ID NO: 80.

In some embodiments, the antibody is selected from the group consistingof a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, an scFv, an Fv,and a full-length IgG. In some embodiments, the antibody is a Fabfragment.

In some embodiments, the antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 97; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 98; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 99; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 103; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

In some embodiments, the antibody comprises:

(i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 98and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99and a light chain comprising the amino acid sequence of SEQ ID NO: 85;

(v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100and a light chain comprising the amino acid sequence of SEQ ID NO: 90;

(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101and a light chain comprising the amino acid sequence of SEQ ID NO: 89;

(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:93;

(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103and a light chain comprising the amino acid sequence of SEQ ID NO: 95;or

(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the equilibrium dissociation constant (K_(D)) ofbinding of the antibody to the transferrin receptor is in a range from10⁻¹¹ M to 10⁻⁶ M. In some embodiments, the antibody does notspecifically bind to the transferrin binding site of the transferrinreceptor and/or the antibody does not inhibit binding of transferrin tothe transferrin receptor. In some embodiments, the antibody iscross-reactive with extracellular epitopes of two or more of a human,non-human primate and rodent transferrin receptor.

Another aspect of the present disclosure relates to a complex comprisingthe antibody covalently linked to a molecular payload. In someembodiments, the molecular payload is a diagnostic agent or atherapeutic agent. In some embodiments, the molecular payload is anoligonucleotide, a polypeptide, or a small molecule. In someembodiments, the antibody and the molecular payload are linked via alinker. In some embodiments, the linker is a cleavable linker. In someembodiments, the linker comprises a valine-citrulline sequence.

Another aspect of the present disclosure relates to a compositioncomprising an antibody or a complex disclosed herein. In someembodiments, the composition further comprises a pharmaceuticallyacceptable carrier.

Yet another aspect of the present disclosure relates to a method ofdelivering a molecular payload to a cell, comprising contacting the cellwith a complex or composition disclosed herein. In some embodiments, thecell is a muscle cell. In some embodiments, the cell is in vitro. Insome embodiments, the cell is in a subject. In some embodiments, thesubject is human.

Another aspect of the present disclosure relates to a method ofdelivering a molecular payload to the muscle of a subject, comprisingadministering to the subject an effective amount of a complex disclosedherein. In some embodiments, the administration is intravenous.

Another aspect of the present disclosure relates to a method of treatinga disease, comprising administering to a subject an effective amount ofa complex or a composition disclosed herein, wherein the molecularpayload is a therapeutic agent. In some embodiments, the disease is amuscle disease and the molecular payload is a drug for treating themuscle disease. In some embodiments, the muscle disease is a rare muscledisease or muscle atrophy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F show binding of humanized anti-TfR Fabs to human TfR1(hTfR1) or cynomolgus monkey TfR1 (cTfR1), as measured by ELISA. FIG. 1Ashows binding of humanized 3M12 variants to hTfR1. FIG. 1B shows bindingof humanized 3M12 variants to cTfR1. FIG. 1C shows binding of humanized3A4 variants to hTfR1. FIG. 1D shows binding of humanized 3A4 variantsto cTfR1. FIG. 1E shows binding of humanized 5H12 variants to hTfR1.FIG. 1F shows binding of humanized 5H12 variants to hTfR1.

FIG. 2 shows the quantified cellular uptake of anti-TfR Fab conjugatesinto rhabdomyosarcoma (RD) cells. The molecular payload in the testedconjugates are DMPK-targeting oligonucleotides and the uptake of theconjugates were facilitated by indicated anti-TfR Fabs. Conjugateshaving a negative control Fab (anti-mouse TfR) or a positive control Fab(anti-human TfR1) are also included this assay. Cells were incubatedwith indicated conjugate at a concentration of 100 nM for 4 hours.Cellular uptake was measured by mean Cypher5e fluorescence.

FIGS. 3A-3F show binding of oligonucleotide-conjugated or unconjugatedhumanized anti-TfR Fabs to human TfR1 (hTfR1) and cynomolgus monkey TfR1(cTfR1), as measured by ELISA. FIG. 3A shows the binding of humanized3M12 variants alone or in conjugates with a DMPK targeting oligo tohTfR1. FIG. 3B shows the binding of humanized 3M12 variants alone or inconjugates with a DMPK targeting oligo to cTfR1. FIG. 3C shows thebinding of humanized 3A4 variants alone or in conjugates with a DMPKtargeting oligo to hTfR1. FIG. 3D shows the binding of humanized 3A4variants alone or in conjugates with a DMPK targeting oligo to cTfR1.FIG. 3E shows the binding of humanized 5H12 variants alone or inconjugates with a DMPK targeting oligo to hTfR1. FIG. 3F shows thebinding of humanized 5H12 variants alone or in conjugates with a DMPKtargeting oligo to cTfR1. The respective EC₅₀ values are also shown.

FIG. 4 shows DMPK expression in RD cells treated with variousconcentrations of conjugates containing the indicated humanized anti-TfRFab antibodies conjugated to a DMPK-targeting oligonucleotide (ASO300).The duration of treatment was 3 days. A50300 delivered usingtransfection agents (labeled “Trans”) was used as control.

FIG. 5 shows the serum stability of the linker used for linking ananti-TfR antibody and a molecular payload (e.g., an oligonucleotide) invarious species over time after intravenous administration.

FIG. 6 shows data illustrating that conjugates containing designatedanti-TfR Fabs (3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3 N54S/VK4)conjugated to a DMD exon-skipping oligonucleotide resulted in enhancedexon skipping compared to the naked DMD exon skipping oligo in DMDpatient myotubes.

FIGS. 7A-7E show in vivo activity of conjugates containing designatedanti-TfR Fabs (control, 3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3N54S/VK4) conjugated to DMPK targeting oligonucleotide in reducing DMPKmRNA expression in mice expressing human TfR1 (hTfR1 knock-in mice).FIG. 7A shows the experimental design (e.g., IV dosage, dosingfrequency). DMPK mRNA levels were measured 14 days post first dose inthe tibialis anterior (FIG. 7B), gastrocnemius (FIG. 7C), heart (FIG.7D), and diaphragm (FIG. 7E), of the mice.

FIG. 8 shows ELISA measurements of binding of anti-TfR Fab 3M12 VH4/Vk3to recombinant human (circles), cynomolgus monkey (squares), mouse(upward triangles), or rat (downward triangles) TfR1 protein, at a rangeof concentrations from 230 pM to 500 nM of the Fab. Measurement resultsshow that the anti-TfR Fab is reactive with human and cynomolgus monkeyTfR1. Binding was not observed to mouse or rat recombinant TfR1. Data isshown as relative fluorescent units normalized to baseline.

FIG. 9 shows results of an ELISA testing the affinity of anti-TfR Fab3M12 VH4/Vk3 to recombinant human TfR1 or TfR2 over a range ofconcentrations from 230 pM to 500 nM of Fab. The data are presented asrelative fluorescence units normalized to baseline. The resultsdemonstrate that the Fab does not bind recombinant human TfR2.

FIG. 10 shows the serum stability of the linker used for linkinganti-TfR Fab 3M12 VH4/Vk3 to a control antisense oligonucleotide over 72hours incubation in PBS or in rat, mouse, cynomolgus monkey or humanserum.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present disclosure, at least in part, is based on the development ofhumanized anti-TfR antibodies, e.g., antibodies listed in Table 3, whichshowed high binding affinity and specificity to human TfR. Also providedare the use of the anti-TfR antibodies and their variants in research,diagnostic/detection, and therapeutic applications. In some embodiments,the anti-TfR antibodies described herein are used for deliveringmolecular payloads (e.g., oligonucleotides, peptides, small molecules)to a target cell or tissue that expresses TfR. In some embodiments, themolecular payload to be delivered is conjugated the anti-TfR antibodiesand delivered to a target cell or tissue that expresses TfR via receptorinternationalization. Exemplary tissues that express TfR and can betargeted using the anti-TfR antibodies described herein include, withoutlimitation: brain, muscle, adrenal, appendix, bone marrow, colon,duodenum, endometrium, esophagus, fat, gall bladder, heart, kidney,liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivarygland, skin, small intestine, spleen, stomach, testis, thyroid, urinarybladder. In some embodiments, such approach has beneficial effects inmuscle cells and for delivering across the blood brain barrier, whichhave been proven challenging. In some aspects, the present disclosureprovides data demonstrating that the anti-TfR antibodies describedherein has superior activity in delivering molecular payload into atarget cell (e.g., a muscle cell), compared with other known anti-TfRantibodies.

As such, the present disclosure also provides complexes comprising anyone of the anti-TfR1 antibodies covalently linked to molecular payloads.In some embodiments, the complexes are particularly useful fordelivering molecular payloads that inhibit the expression or activity oftarget genes in muscle cells, e.g., in a subject having or suspected ofhaving a rare muscle disease or muscle atrophy (e.g., as listed in Table6). In some embodiments, the complexes are particularly useful fordelivering drugs to the brain for treating a neurological disease (e.g.,as listed in Table 7).

Further aspects of the disclosure, including a description of definedterms, are provided below.

Definitions

Administering: As used herein, the terms “administering” or“administration” means to provide a complex to a subject in a mannerthat is physiologically and/or (e.g., and) pharmacologically useful(e.g., to treat a condition in the subject).

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, orless in either direction (greater than or less than) of the statedreference value unless otherwise stated or otherwise evident from thecontext (except where such number would exceed 100% of a possiblevalue).

Antibody: As used herein, the term “antibody” refers to a polypeptidethat includes at least one immunoglobulin variable domain or at leastone antigenic determinant, e.g., paratope that specifically binds to anantigen. In some embodiments, an antibody is a full-length antibody. Insome embodiments, an antibody is a chimeric antibody. In someembodiments, an antibody is a humanized antibody. However, in someembodiments, an antibody is a Fab fragment, a Fab′ fragment, a F(ab′)2fragment, a Fv fragment or a scFv fragment. In some embodiments, anantibody is a nanobody derived from a camelid antibody or a nanobodyderived from shark antibody. In some embodiments, an antibody is adiabody. In some embodiments, an antibody comprises a framework having ahuman germline sequence. In another embodiment, an antibody comprises aheavy chain constant domain selected from the group consisting of IgG,IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, andIgE constant domains. In some embodiments, an antibody comprises a heavy(H) chain variable region (abbreviated herein as VH), and/or (e.g., and)a light (L) chain variable region (abbreviated herein as VL). In someembodiments, an antibody comprises a constant domain, e.g., an Fcregion. An immunoglobulin constant domain refers to a heavy or lightchain constant domain. Human IgG heavy chain and light chain constantdomain amino acid sequences and their functional variations are known.With respect to the heavy chain, in some embodiments, the heavy chain ofan antibody described herein can be an alpha (α), delta (Δ), epsilon(ε), gamma (γ) or mu (μ) heavy chain. In some embodiments, the heavychain of an antibody described herein can comprise a human alpha (α),delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In a particularembodiment, an antibody described herein comprises a human gamma 1 CHLCH2, and/or (e.g., and) CH3 domain. In some embodiments, the amino acidsequence of the VH domain comprises the amino acid sequence of a humangamma (γ) heavy chain constant region, such as any known in the art.Non-limiting examples of human constant region sequences have beendescribed in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A etal., (1991) supra. In some embodiments, the VH domain comprises an aminoacid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or atleast 99% identical to any of the variable chain constant regionsprovided herein. In some embodiments, an antibody is modified, e.g.,modified via glycosylation, phosphorylation, sumoylation, and/or (e.g.,and) methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecule are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, an antibody is a constructthat comprises a polypeptide comprising one or more antigen bindingfragments of the disclosure linked to a linker polypeptide or animmunoglobulin constant domain. Linker polypeptides comprise two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Examples of linker polypeptides have beenreported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).Still further, an antibody may be part of a larger immunoadhesionmolecule, formed by covalent or noncovalent association of the antibodyor antibody portion with one or more other proteins or peptides.Examples of such immunoadhesion molecules include use of thestreptavidin core region to make a tetrameric scFv molecule (Kipriyanov,S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and useof a cysteine residue, a marker peptide and a C-terminal polyhistidinetag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M.,et al. (1994) Mol. Immunol. 31:1047-1058).

CDR: As used herein, the term “CDR” refers to the complementaritydetermining region within antibody variable sequences. A typicalantibody molecule comprises a heavy chain variable region (VH) and alight chain variable region (VL), which are usually involved in antigenbinding. The VH and VL regions can be further subdivided into regions ofhypervariability, also known as “complementarity determining regions”(“CDR”), interspersed with regions that are more conserved, which areknown as “framework regions” (“FR”). Each VH and VL is typicallycomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The extent of the framework region and CDRs can be preciselyidentified using methodology known in the art, for example, by the Kabatdefinition, the IMGT definition, the Chothia definition, the AbMdefinition, and/or (e.g., and) the contact definition, all of which arewell known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; IMGT®, theinternational ImMunoGeneTics information System® http://www.imgt.org,Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M.et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., NucleicAcids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res.,31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006(2004) [Epub], 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic AcidsRes., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res.,37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res.,43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J.Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143(2004). See also hgmp.mrc.ac.uk and bioinforg.uk/abs. As used herein, aCDR may refer to the CDR defined by any method known in the art. Twoantibodies having the same CDR means that the two antibodies have thesame amino acid sequence of that CDR as determined by the same method,for example, the IMGT definition.

There are three CDRs in each of the variable regions of the heavy chainand the light chain, which are designated CDR1, CDR2 and CDR3, for eachof the variable regions. The term “CDR set” as used herein refers to agroup of three CDRs that occur in a single variable region capable ofbinding the antigen. The exact boundaries of these CDRs have beendefined differently according to different systems. The system describedby Kabat (Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. (1987) and (1991)) notonly provides an unambiguous residue numbering system applicable to anyvariable region of an antibody, but also provides precise residueboundaries defining the three CDRs. These CDRs may be referred to asKabat CDRs. Sub-portions of CDRs may be designated as L1, L2 and L3 orH1, H2 and H3 where the “L” and the “H” designates the light chain andthe heavy chains regions, respectively. These regions may be referred toas Chothia CDRs, which have boundaries that overlap with Kabat CDRs.Other boundaries defining CDRs overlapping with the Kabat CDRs have beendescribed by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J MolBiol 262(5):732-45 (1996)). Still other CDR boundary definitions may notstrictly follow one of the above systems, but will nonetheless overlapwith the Kabat CDRs, although they may be shortened or lengthened inlight of prediction or experimental findings that particular residues orgroups of residues or even entire CDRs do not significantly impactantigen binding. The methods used herein may utilize CDRs definedaccording to any of these systems. Examples of CDR definition systemsare provided in Table 1.

TABLE 1 CDR Definitions IMGT¹ Kabat² Chothia³ CDR-H1  27-38 31-35 26-32CDR-H2  56-65 50-65 53-55 CDR-H3 105-116/117 95-102 96-101 CDR-L1  27-3824-34 26-32 CDR-L2  56-65 50-56 50-52 CDR-L3 105-116/117 89-97 91-96¹IMGT ®, the international ImMunoGeneTics information system ®,imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999)²Kabat et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242 ³Chothia et al., J. Mol. Biol. 196:901-917(1987))

CDR-grafted antibody: The term “CDR-grafted antibody” refers toantibodies which comprise heavy and light chain variable regionsequences from one species but in which the sequences of one or more ofthe CDR regions of VH and/or (e.g., and) VL are replaced with CDRsequences of another species, such as antibodies having murine heavy andlight chain variable regions in which one or more of the murine CDRs(e.g., CDR3) has been replaced with human CDR sequences.

Chimeric antibody: The term “chimeric antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from onespecies and constant region sequences from another species, such asantibodies having murine heavy and light chain variable regions linkedto human constant regions.

Complementary: As used herein, the term “complementary” refers to thecapacity for precise pairing between two nucleotides or two sets ofnucleotides. In particular, complementary is a term that characterizesan extent of hydrogen bond pairing that brings about binding between twonucleotides or two sets of nucleotides. For example, if a base at oneposition of an oligonucleotide is capable of hydrogen bonding with abase at the corresponding position of a target nucleic acid (e.g., anmRNA), then the bases are considered to be complementary to each otherat that position. Base pairings may include both canonical Watson-Crickbase pairing and non-Watson-Crick base pairing (e.g., Wobble basepairing and Hoogsteen base pairing). For example, in some embodiments,for complementary base pairings, adenosine-type bases (A) arecomplementary to thymidine-type bases (T) or uracil-type bases (U), thatcytosine-type bases (C) are complementary to guanosine-type bases (G),and that universal bases such as 3-nitropyrrole or 5-nitroindole canhybridize to and are considered complementary to any A, C, U, or T.Inosine (I) has also been considered in the art to be a universal baseand is considered complementary to any A, C, U or T.

Conservative amino acid substitution: As used herein, a “conservativeamino acid substitution” refers to an amino acid substitution that doesnot alter the relative charge or size characteristics of the protein inwhich the amino acid substitution is made. Variants can be preparedaccording to methods for altering polypeptide sequence known to one ofordinary skill in the art such as are found in references which compilesuch methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook,et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.Conservative substitutions of amino acids include substitutions madeamongst amino acids within the following groups: (a) M, I, L, V; (b) F,Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Covalently linked: As used herein, the term “covalently linked” refersto a characteristic of two or more molecules being linked together viaat least one covalent bond. In some embodiments, two molecules can becovalently linked together by a single bond, e.g., a disulfide bond ordisulfide bridge, that serves as a linker between the molecules.However, in some embodiments, two or more molecules can be covalentlylinked together via a molecule that serves as a linker that joins thetwo or more molecules together through multiple covalent bonds. In someembodiments, a linker may be a cleavable linker. However, in someembodiments, a linker may be a non-cleavable linker.

Cross-reactive: As used herein and in the context of a targeting agent(e.g., antibody), the term “cross-reactive,” refers to a property of theagent being capable of specifically binding to more than one antigen ofa similar type or class (e.g., antigens of multiple homologs, paralogs,or orthologs) with similar affinity or avidity. For example, in someembodiments, an antibody that is cross-reactive against human andnon-human primate antigens of a similar type or class (e.g., a humantransferrin receptor and non-human primate transferrin receptor) iscapable of binding to the human antigen and non-human primate antigenswith a similar affinity or avidity. In some embodiments, an antibody iscross-reactive against a human antigen and a rodent antigen of a similartype or class. In some embodiments, an antibody is cross-reactiveagainst a rodent antigen and a non-human primate antigen of a similartype or class. In some embodiments, an antibody is cross-reactiveagainst a human antigen, a non-human primate antigen, and a rodentantigen of a similar type or class.

Framework: As used herein, the term “framework” or “framework sequence”refers to the remaining sequences of a variable region minus the CDRs.Because the exact definition of a CDR sequence can be determined bydifferent systems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, CDR-L2,and CDR-L3 of light chain and CDR-H1, CDR-H2, and CDR-H3 of heavy chain)also divide the framework regions on the light chain and the heavy chaininto four sub-regions (FR1, FR2, FR3 and FR4) on each chain, in whichCDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, andCDR3 between FR3 and FR4. Without specifying the particular sub-regionsas FR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FRs within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region. Human heavychain and light chain acceptor sequences are known in the art. In oneembodiment, the acceptor sequences known in the art may be used in theantibodies disclosed herein.

Human antibody: The term “human antibody”, as used herein, is intendedto include antibodies having variable and constant regions derived fromhuman germline immunoglobulin sequences. The human antibodies of thedisclosure may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

Humanized antibody: The term “humanized antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theVH and/or (e.g., and) VL sequence has been altered to be more“human-like”, i.e., more similar to human germline variable sequences.One type of humanized antibody is a CDR-grafted antibody, in which humanCDR sequences are introduced into non-human VH and VL sequences toreplace the corresponding nonhuman CDR sequences. In one embodiment,humanized anti-TfR antibodies and antigen binding portions are provided.Such antibodies may be generated by obtaining murine anti-transferrinreceptor monoclonal antibodies using traditional hybridoma technologyfollowed by humanization using in vitro genetic engineering, such asthose disclosed in Kasaian et al PCT publication No. WO 2005/123126 A2.

Isolated antibody: An “isolated antibody”, as used herein, is intendedto refer to an antibody that is substantially free of other antibodieshaving different antigenic specificities (e.g., an isolated antibodythat specifically binds transferrin receptor is substantially free ofantibodies that specifically bind antigens other than transferrinreceptor). An isolated antibody that specifically binds transferrinreceptor complex may, however, have cross-reactivity to other antigens,such as transferrin receptor molecules from other species. Moreover, anisolated antibody may be substantially free of other cellular materialand/or (e.g., and) chemicals.

Molecular payload: As used herein, the term “molecular payload” refersto a molecule or species that functions to modulate a biologicaloutcome. In some embodiments, a molecular payload is linked to, orotherwise associated with an anti-TfR antibody. In some embodiments, themolecular payload is a small molecule, a protein, a peptide, a nucleicacid, or an oligonucleotide. In some embodiments, the molecular payloadfunctions to modulate the transcription of a DNA sequence, to modulatethe expression of a protein, or to modulate the activity of a protein.In some embodiments, the molecular payload is an oligonucleotide thatcomprises a strand having a region of complementarity to a target gene.

Oligonucleotide: As used herein, the term “oligonucleotide” refers to anoligomeric nucleic acid compound of up to 200 nucleotides in length.Examples of oligonucleotides include, but are not limited to, RNAioligonucleotides (e.g., siRNAs, shRNAs), microRNAs, gapmers, mixmers,phosphorodiamidite morpholinos, peptide nucleic acids, aptamers, guidenucleic acids (e.g., Cas9 guide RNAs), etc. Oligonucleotides may besingle-stranded or double-stranded. In some embodiments, anoligonucleotide may comprise one or more modified nucleotides (e.g.2′-O-methyl sugar modifications, purine or pyrimidine modifications). Insome embodiments, an oligonucleotide may comprise one or more modifiedinternucleotide linkage. In some embodiments, an oligonucleotide maycomprise one or more phosphorothioate linkages, which may be in the Rpor Sp stereochemical conformation.

Recombinant antibody: The term “recombinant human antibody”, as usedherein, is intended to include all human antibodies that are prepared,expressed, created or isolated by recombinant means, such as antibodiesexpressed using a recombinant expression vector transfected into a hostcell (described in more details in this disclosure), antibodies isolatedfrom a recombinant, combinatorial human antibody library (Hoogenboom H.R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002)Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002)BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) ImmunologyToday 21:371-378), antibodies isolated from an animal (e.g., a mouse)that is transgenic for human immunoglobulin genes (see e.g., Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., andGreen L. L. (2002) Current Opinion in Biotechnology 13:593-597; LittleM. et al (2000) Immunology Today 21:364-370) or antibodies prepared,expressed, created or isolated by any other means that involves splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies are subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo. One embodiment of the disclosure provides fully human antibodiescapable of binding human transferrin receptor which can be generatedusing techniques well known in the art, such as, but not limited to,using human Ig phage libraries such as those disclosed in Jermutus etal., PCT publication No. WO 2005/007699 A2.

Region of complementarity: As used herein, the term “region ofcomplementarity” refers to a nucleotide sequence, e.g., of anoligonucleotide, that is sufficiently complementary to a cognatenucleotide sequence, e.g., of a target nucleic acid, such that the twonucleotide sequences are capable of annealing to one another underphysiological conditions (e.g., in a cell). In some embodiments, aregion of complementarity is fully complementary to a cognate nucleotidesequence of target nucleic acid. However, in some embodiments, a regionof complementarity is partially complementary to a cognate nucleotidesequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99%complementarity). In some embodiments, a region of complementaritycontains 1, 2, 3, or 4 mismatches compared with a cognate nucleotidesequence of a target nucleic acid.

Specifically binds: As used herein, the term “specifically binds” refersto the ability of a molecule to bind to a binding partner with a degreeof affinity or avidity that enables the molecule to be used todistinguish the binding partner from an appropriate control in a bindingassay or other binding context. With respect to an antibody, the term,“specifically binds”, refers to the ability of the antibody to bind to aspecific antigen with a degree of affinity or avidity, compared with anappropriate reference antigen or antigens, that enables the antibody tobe used to distinguish the specific antigen from others, e.g., to anextent that permits preferential targeting to certain cells, e.g.,muscle cells, through binding to the antigen, as described herein. Insome embodiments, an antibody specifically binds to a target if theantibody has a K_(D) for binding the target of at least about 10⁻⁴ M,10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³M, or less. In some embodiments, an antibody specifically binds to thetransferrin receptor.

Subject: As used herein, the term “subject” refers to a mammal. In someembodiments, a subject is non-human primate, or rodent. In someembodiments, a subject is a human. In some embodiments, a subject is apatient, e.g., a human patient that has or is suspected of having adisease. In some embodiments, the subject is a human patient who has oris suspected of having a disease resulting from adisease-associated-repeat expansion, e.g., in a DMPK allele.

Transferrin receptor: As used herein, the term, “transferrin receptor”(also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalizingcell surface receptor that binds transferrin to facilitate iron uptakeby endocytosis. In some embodiments, a transferrin receptor may be ofhuman (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042) origin.In addition, multiple human transcript variants have been characterizedthat encoded different isoforms of the receptor (e.g., as annotatedunder GenBank RefSeq Accession Numbers: NP_001121620.1, NP_003225.2,NP_001300894.1, and NP_001300895.1).

An example human transferrin receptor amino acid sequence, correspondingto NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1,Homo sapiens) is as follows:

(SEQ ID NO: 105)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDI DNEF.

An example non-human primate transferrin receptor amino acid sequence,corresponding to NCBI sequence NP_001244232.1 (transferrin receptorprotein 1, Macaca mulatta) is as follows:

(SEQ ID NO: 106)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVW DIDNEF

An example non-human primate transferrin receptor amino acid sequence,corresponding to NCBI sequence XP_005545315.1 (transferrin receptorprotein 1, Macaca fascicularis) is as follows:

(SEQ ID NO: 107)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVW DIDNEF.

An example mouse transferrin receptor amino acid sequence, correspondingto NCBI sequence NP_001344227.1 (transferrin receptor protein 1, Musmusculus) is as follows:

(SEQ ID NO: 108)MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNID NEF.

2′-modified nucleoside: As used herein, the terms “2′-modifiednucleoside” and “2′-modified ribonucleoside” are used interchangeablyand refer to a nucleoside having a sugar moiety modified at the 2′position. In some embodiments, the 2′-modified nucleoside is a 2′-4′bicyclic nucleoside, where the 2′ and 4′ positions of the sugar arebridged (e.g., via a methylene, an ethylene, or a (S)-constrained ethylbridge). In some embodiments, the 2′-modified nucleoside is anon-bicyclic 2′-modified nucleoside, e.g., where the 2′ position of thesugar moiety is substituted. Non-limiting examples of 2′-modifiednucleosides include: 2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE),2′-O—N-methylacetamido (2′-O-NMA), locked nucleic acid (LNA,methylene-bridged nucleic acid), ethylene-bridged nucleic acid (ENA),and (S)-constrained ethyl-bridged nucleic acid (cEt). In someembodiments, the 2′-modified nucleosides described herein arehigh-affinity modified nucleotides and oligonucleotides comprising the2′-modified nucleotides have increased affinity to a target sequences,relative to an unmodified oligonucleotide. Examples of structures of2′-modified nucleosides are provided below:

II. Anti-TfR Antibodies

In some embodiments, agents binding to transferrin receptor, e.g.,anti-TfR antibodies, are capable of targeting muscle cell and/or (e.g.,and) mediate the transportation of an agent across the blood brainbarrier. Transferrin receptors are internalizing cell surface receptorsthat transport transferrin across the cellular membrane and participatein the regulation and homeostasis of intracellular iron levels. Someaspects of the disclosure provide transferrin receptor binding proteins,which are capable of binding to transferrin receptor. Antibodies thatbind, e.g. specifically bind, to a transferrin receptor may beinternalized into the cell, e.g. through receptor-mediated endocytosis,upon binding to a transferrin receptor.

Provided herein, in some aspects, are humanized antibodies that bind totransferrin receptor with high specificity and affinity. In someembodiments, the humanized anti-TfR antibody described hereinspecifically binds to any extracellular epitope of a transferrinreceptor or an epitope that becomes exposed to an antibody. In someembodiments, the humanized anti-TfR antibodies provided herein bindspecifically to transferrin receptor from human, non-human primates,mouse, rat, etc. In some embodiments, the humanized anti-TfR antibodiesprovided herein bind to human transferrin receptor. In some embodiments,the humanized anti-TfR antibody described herein binds to an amino acidsegment of a human or non-human primate transferrin receptor, asprovided in SEQ ID NOs: 105-108. In some embodiments, the humanizedanti-TfR antibody described herein binds to an amino acid segmentcorresponding to amino acids 90-96 of a human transferrin receptor asset forth in SEQ ID NO: 105, which is not in the apical domain of thetransferrin receptor. In some embodiments, the humanized anti-TfRantibodies described herein binds to TfR1 but does not bind to TfR2.

In some embodiments, the anti-TfR antibody described herein (e.g., 3M12and humanized variants) bind an epitope in TfR1, wherein the epitopecomprises residues in amino acids 258-291 and/or amino acids 358-381 ofSEQ ID NO: 105. In some embodiments, the anti-TfR antibodies (e.g., 3M12and humanized variants) described herein bind an epitope comprisingresidues in amino acids amino acids 258-291 and amino acids 358-381 ofSEQ ID NO: 105. In some embodiments, the anti-TfR antibodies describedherein (e.g., 3M12 and humanized variants) bind an epitope comprisingone or more of residues K261, 5273, Y282, T362, S368, S370, and K371 ofhuman TfR1 as set forth in SEQ ID NO: 105. In some embodiments, theanti-TfR antibodies described herein (e.g., 3M12 and humanized variants)bind an epitope comprising residues K261, S273, Y282, T362, S368, S370,and K371 of human TfR1 as set forth in SEQ ID NO: 105.

In some embodiments, an anti-TFR antibody specifically binds a TfR1(e.g., a human or non-human primate TfR1) with binding affinity (e.g.,as indicated by Kd) of at least about 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M,10⁻⁸ M, 10⁻⁹M, 10⁻¹⁰ M, 10⁻¹¹M, 10⁻¹² M, 10⁻¹³ M, or less. In someembodiments, the anti-TfR antibodies described herein binds to TfR1 witha KD of sub-nanomolar range. In some embodiments, the anti-TfRantibodies described herein selectively binds to transferrin receptor 1(TfR1) but do not bind to transferrin receptor 2 (TfR2). In someembodiments, the anti-TfR antibodies described herein binds to humanTfR1 and cyno TfR1 (e.g., with a Kd of 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M,10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, or less), but does not bind to a mouse TfR1.The affinity and binding kinetics of the anti-TfR antibody can be testedusing any suitable method including but not limited to biosensortechnology (e.g., OCTET or BIACORE). In some embodiments, binding of anyone of the anti-TfR antibody described herein does not complete with orinhibit transferrin binding to the TfR1. In some embodiments, binding ofany one of the anti-TfR antibody described herein does not complete withor inhibit HFE-beta-2-microglobulin binding to the TfR1.

The anti-TfR antibodies described herein are humanized antibodies. TheCDR and variable region amino acid sequences of the mouse monoclonalanti-TfR antibody from which the humanized anti-TfR antibodies describedherein are derived are provided in Table 2.

TABLE 2 Mouse Monoclonal Anti-TfR Antibodies No. Ab system IMGT KabatChothia 3-A4 CDR- GFNIKDDY DDYMY GFNIKDD H1 (SEQ ID NO: 1)(SEQ ID NO: 7) (SEQ ID NO: 12) CDR- IDPENGDT WIDPENGDTEYASKFQD ENG H2(SEQ ID NO: 2) (SEQ ID NO: 8) (SEQ ID NO: 13) CDR- TLWLRRGLDY WLRRGLDYLRRGLD H3 (SEQ ID NO: 3) (SEQ ID NO: 9) (SEQ ID NO: 14) CDR- KSLLHSNGYTYRSSKSLLHSNGYTYLF SKSLLHSNGYTY L1 (SEQ ID NO: 4) (SEQ ID NO: 10)(SEQ ID NO: 15) CDR- RMS RMSNLAS RMS L2 (SEQ ID NO: 5) (SEQ ID NO: 11)(SEQ ID NO: 5) CDR- MQHLEYPFT MQHLEYPFT HLEYPF L3 (SEQ ID NO: 6)(SEQ ID NO: 6) (SEQ ID NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPENGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 17) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18)3-A4 CDR- GFNIKDDY DDYMY GFNIKDD N54T* H1 (SEQ ID NO: 1) (SEQ ID NO: 7)(SEQ ID NO: 12) CDR- IDPETGDT WIDPETGDTEYASKFQD ETG H2 (SEQ ID NO: 19)(SEQ ID NO: 20) (SEQ ID NO: 21) CDR- TLWLRRGLDY WLRRGLDY LRRGLD H3(SEQ ID NO: 3) (SEQ ID NO: 9) (SEQ ID NO: 14) CDR- KSLLHSNGYTYRSSKSLLHSNGYTYLF SKSLLHSNGYTY L1 (SEQ ID NO: 4) (SEQ ID NO: 10)(SEQ ID NO: 15) CDR- RMS RMSNLAS RMS L2 (SEQ ID NO: 5) (SEQ ID NO: 11)(SEQ ID NO: 5) CDR- MQHLEYPFT MQHLEYPFT HLEYPF L3 (SEQ ID NO: 6)(SEQ ID NO: 6) (SEQ ID NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPETGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 22) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18)3-A4 CDR- GFNIKDDY DDYMY GFNIKDD N54S* H1 (SEQ ID NO: 1) (SEQ ID NO: 7)(SEQ ID NO: 12) CDR- IDPESGDT WIDPESGDTEYASKFQD ESG H2 (SEQ ID NO: 23)(SEQ ID NO: 24) (SEQ ID NO: 25) CDR- TLWLRRGLDY WLRRGLDY LRRGLD H3(SEQ ID NO: 3) (SEQ ID NO: 9) (SEQ ID NO: 14) CDR- KSLLHSNGYTYRSSKSLLHSNGYTYLF SKSLLHSNGYTY L1 (SEQ ID NO: 4) (SEQ ID NO: 10)(SEQ ID NO: 15) CDR- RMS RMSNLAS RMS L2 (SEQ ID NO: 5) (SEQ ID NO: 11)(SEQ ID NO: 5) CDR- MQHLEYPFT MQHLEYPFT HLEYPF L3 (SEQ ID NO: 6)(SEQ ID NO: 6) (SEQ ID NO: 16) VHEVQLQQSGAELVRPGASVKLSCTASGFNIKDDYMYWVKQRPEQGLEWIGWIDPESGDTEYASKFQDKATVTADTSSNTAYLQLSSLTSEDTAVYYCTLWLRRGLDYWGQGTSVTVSS (SEQ ID NO: 26) VLDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGYTYLFWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGGGTKLEIK (SEQ ID NO: 18)3-M12 CDR- GYSITSGYY SGYYWN GYSITSGY H1 (SEQ ID NO: 27) (SEQ ID NO: 33)(SEQ ID NO: 38) CDR- ITFDGAN YITFDGANNYNPSLKN FDG H2 (SEQ ID NO: 28)(SEQ ID NO: 34) (SEQ ID NO: 39) CDR- TRSSYDYDVLDY SSYDYDVLDY SYDYDVLD H3(SEQ ID NO: 29) (SEQ ID NO: 35) (SEQ ID NO: 40) CDR- QDISNF RASQDISNFLNSQDISNF L1 (SEQ ID NO: 30) (SEQ ID NO: 36) (SEQ ID NO: 41) CDR- YTSYTSRLHS YTS L2 (SEQ ID NO: 31) (SEQ ID NO: 37) (SEQ ID NO: 31) CDR-QQGHTLPYT QQGHTLPYT GHTLPY L3 (SEQ ID NO: 32) (SEQ ID NO: 32)(SEQ ID NO: 42) VHDVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYITFDGANNYNPSLKNRISITRDTSKNQFFLKLTSVTTEDTATYYCTRSSYDYDVLDYWGQGTTLTVSS (SEQ ID NO: 43) VLDIQMTQTTSSLSASLGDRVTISCRASQDISNFLNWYQQRPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDFSLTVSNLEOEDIATYFCOOGHTLPYTFGGGTKLEIK (SEQ ID NO: 44) 5-H12CDR- GYSFTDYC DYCIN) GYSFTDY H1 (SEQ ID NO: 45) (SEQ ID NO: 51(SEQ ID NO: 56) CDR- IYPGSGNT WIYPGSGNTRYSERFKG GSG H2 (SEQ ID NO: 46)(SEQ ID NO: 52) (SEQ ID NO: 57) CDR- AREDYYPYHGMDY EDYYPYHGMDY DYYPYHGMDH3 (SEQ ID NO: 47) (SEQ ID NO: 53) (SEQ ID NO: 58) CDR- ESVDGYDNSFRASESVDGYDNSFMH SESVDGYDNSF L1 (SEQ ID NO: 48) (SEQ ID NO: 54)(SEQ ID NO: 59) CDR- RAS RASNLES RAS L2 (SEQ ID NO: 49) (SEQ ID NO: 55)(SEQ ID NO: 49) CDR- QQSSEDPWT QQSSEDPWT) SSEDPW L3 (SEQ ID NO: 50)(SEQ ID NO: 50 (SEQ ID NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYCINWVNQRPGQGLEWIGWIYPGSGNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 61) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62)5-H12 CDR- GYSFTDYY DYYIN GYSFTDY C33Y* H1 (SEQ ID NO: 63)(SEQ ID NO: 64) (SEQ ID NO: 56) CDR- IYPGSGNT WIYPGSGNTRYSERFKG GSG H2(SEQ ID NO: 46) (SEQ ID NO: 52) (SEQ ID NO: 57) CDR- AREDYYPYHGMDYEDYYPYHGMDY DYYPYHGMD H3 (SEQ ID NO: 47) (SEQ ID NO: 53) (SEQ ID NO: 58)CDR- ESVDGYDNSF RASESVDGYDNSFMH SESVDGYDNSF Ll (SEQ ID NO: 48)(SEQ ID NO: 54) (SEQ ID NO: 59) CDR- RAS RASNLES RAS L2 (SEQ ID NO: 49)(SEQ ID NO: 55) (SEQ ID NO: 49) CDR- QQSSEDPWT QQSSEDPWT SSEDPW) L3(SEQ ID NO: 50) (SEQ ID NO: 50) (SEQ ID NO: 60 VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYYINWVNQRPGQGLEWIGWIYPGSGNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 65) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62)5-H12 CDR- GYSFTDYD DYDIN GYSFTDY C33D* H1 (SEQ ID NO: 66)(SEQ ID NO: 67) (SEQ ID NO: 56) CDR- IYPGSGNT WIYPGSGNTRYSERFKG GSG H2(SEQ ID NO: 46) (SEQ ID NO: 52) (SEQ ID NO: 57) CDR- AREDYYPYHGMDYEDYYPYHGMDY DYYPYHGMD H3 (SEQ ID NO: 47) (SEQ ID NO: 53) (SEQ ID NO: 58)CDR- ESVDGYDNSF RASESVDGYDNSFMH SESVDGYDNSF L1 (SEQ ID NO: 48)(SEQ ID NO: 54) (SEQ ID NO: 59) CDR- RAS RASNLES RAS L2 (SEQ ID NO: 49)(SEQ ID NO: 55) (SEQ ID NO: 49) CDR- QQSSEDPWT QQSSEDPWT SSEDPW L3(SEQ ID NO: 50) (SEQ ID NO: 50) (SEQ ID NO: 60) VHQIQLQQSGPELVRPGASVKISCKASGYSFTDYDINWVNQRPGQGLEWIGWIYPGSGNTRYSERFKGKATLTVDTSSNTAYMQLSSLTSEDSAVYFCAREDYYPYHGMDYWGQGTSVTVSS (SEQ ID NO: 68) VLDIVLTQSPTSLAVSLGQRATISCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGIPARFSGSGSRTDFTLTINPVEAADVATYYCQQSSEDPWTFGGGTKLEIK (SEQ ID NO: 62)*mutation positions are according to Kabat numbering of the respectiveVH sequences containing the mutations

In some embodiments, the anti-TfR antibody of the present disclosure isa humanized variant of any one of the anti-TfR antibodies provided inTable 2. In some embodiments, the anti-TfR antibody of the presentdisclosure comprises a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, a CDR-L2,and a CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 in anyone of the anti-TfR antibodies provided in Table 2, and comprises ahumanized heavy chain variable region and/or (e.g., and) a humanizedlight chain variable region.

Humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some embodiments, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,six) which are altered with respect to the original antibody, which arealso termed one or more CDRs derived from one or more CDRs from theoriginal antibody. Humanized antibodies may also involve affinitymaturation.

Humanized antibodies and methods of making them are known, e.g., asdescribed in Almagro et al., Front. Biosci. 13:1619-1633 (2008);Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'lAcad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34(2005); Padlan et al., Mol. Immunol. 28:489-498 (1991); Dall'Acqua etal., Methods 36:43-60 (2005); Osbourn et al., Methods 36:61-68 (2005);and Klimka et al., Br. J. Cancer, 83:252-260 (2000), the contents of allof which are incorporated herein by reference. Human framework regionsthat may be used for humanization are described in e.g., Sims et al. J.Immunol. 151:2296 (1993); Carter et al., Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993); Almagro etal., Front. Biosci. 13:1619-1633 (2008)); Baca et al., J. Biol. Chem.272:10678-10684 (1997); and Rosok et al., J Biol. Chem. 271:22611-22618(1996), the contents of all of which are incorporated herein byreference.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising one or more amino acidvariations (e.g., in the VH framework region) as compared with any oneof the VHs listed in Table 2, and/or (e.g., and) a humanized VLcomprising one or more amino acid variations (e.g., in the VL frameworkregion) as compared with any one of the VLs listed in Table 2.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH containing no more than 25 aminoacid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VH of any ofthe anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ ID NOs:17, 22, 26, 43, 61, 65, and 68). Alternatively or in addition (e.g., inaddition), the humanized anti-TfR antibody of the present disclosurecomprises a humanized VL containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL of any oneof the anti-TfR antibodies listed in Table 2 (e.g., any one of SEQ IDNOs: 18, 44, and 62).

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%)identical in the framework regions to the VH of any of the anti-TfRantibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 17, 22, 26,43, 61, 65, and 68). Alternatively or in addition (e.g., in addition),In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VL comprising an amino acid sequencethat is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%)identical in the framework regions to the VL of any of the anti-TfRantibodies listed in Table 2 (e.g., any one of SEQ ID NOs: 18, 44, and62).

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 1 (according to the IMGT definition system),a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19,or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3having the amino acid sequence of SEQ ID NO: 3 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VH as set forth in SEQ ID NO: 17,SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or in addition (e.g., inaddition), the anti-TfR antibody of the present disclosure comprises ahumanized VL comprising a CDR-L1 having the amino acid sequence of SEQID NO: 4 (according to the IMGT definition system), a CDR-L2 having theamino acid sequence of SEQ ID NO: 5 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 6(according to the IMGT definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VL as setforth in SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 1 (according to the IMGT definition system),a CDR-H2 having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 19,or SEQ ID NO: 23 (according to the IMGT definition system), a CDR-H3having the amino acid sequence of SEQ ID NO: 3 (according to the IMGTdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VH as set forthin SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 4 (according to the IMGTdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 5 (according to the IMGT definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 6 (according to the IMGT definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VL as set forth in anyone of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 7 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), aCDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to theKabat definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VH as set forthin SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 10 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 11 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 6 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 7 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8, SEQ IDNO: 20, or SEQ ID NO: 24 (according to the Kabat definition system), aCDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to theKabat definition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%,95%, 98%, or 99%) identical in the framework regions to the VH as setforth in SEQ ID NO: 17, SEQ ID NO: 22, or SEQ ID NO: 26. Alternativelyor in addition (e.g., in addition), the humanized anti-TfR antibody ofthe present disclosure comprises a humanized VL comprising a CDR-L1having the amino acid sequence of SEQ ID NO: 10 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 11 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 6 (according to the Kabatdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin any one of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 12 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQID NO: 21, or SEQ ID NO: 25 (according to the Chothia definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14(according to the Chothia definition system), and containing no morethan 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid variation) in the framework regions as compared with the VHas set forth in SEQ ID NO: 17, SEQ ID NO: 22 or SEQ ID NO: 26.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 15 (according to theChothia definition system), a CDR-L2 having the amino acid sequence ofSEQ ID NO: 5 (according to the Chothia definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 16 (according to theChothia definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL as set forthin SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 12 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 13, SEQID NO: 21, or SEQ ID NO: 25 (according to the Chothia definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 14(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: SEQ ID NO: 17, SEQ ID NO: 22 or SEQID NO: 26. Alternatively or in addition (e.g., in addition), theanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 15(according to the Chothia definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 5 (according to the Chothia definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 16(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in any one of SEQ ID NO: 18.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 27 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 29 (according to the IMGT definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 30 (according to theIMGT definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 31 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 32 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 27 (according to the IMGT definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 28(according to the IMGT definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 29 (according to the IMGT definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 30(according to the IMGT definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 31 (according to the IMGT definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32(according to the IMGT definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 33 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 35 (according to the Kabat definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 36 (according to theKabat definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 37 (according to the Kabat definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 32 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 33 (according to the Kabat definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 34(according to the Kabat definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 35 (according to the Kabat definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 36(according to the Kabat definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 37 (according to the Kabat definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 32(according to the Kabat definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 38 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 40 (according to the Chothia definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 43.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 41 (according to theChothia definition system), a CDR-L2 having the amino acid sequence ofSEQ ID NO: 31 (according to the Chothia definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 42 (according to theChothia definition system), and containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) in the framework regions as compared with the VL as set forthin SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 38 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 39(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 40 (according to the Chothia definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 43. Alternatively or in addition (e.g., in addition), the humanizedanti-TfR antibody of the present disclosure comprises a humanized VLcomprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 41(according to the Chothia definition system), a CDR-L2 having the aminoacid sequence of SEQ ID NO: 31 (according to the Chothia definitionsystem), and a CDR-L3 having the amino acid sequence of SEQ ID NO: 42(according to the Chothia definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VL as set forth in SEQ ID NO: 44.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66(according to the IMGT definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 46 (according to the IMGT definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47(according to the IMGT definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VH as setforth in SEQ ID NO: 61, SEQ ID NO: 65, or SEQ ID NO: 68. Alternativelyor in addition (e.g., in addition), the humanized anti-TfR antibody ofthe present disclosure comprises a humanized VL comprising a CDR-L1having the amino acid sequence of SEQ ID NO: 48 (according to the IMGTdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 49 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the IMGTdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 45, SEQ ID NO: 63, or SEQ ID NO: 66(according to the IMGT definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 46 (according to the IMGT definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 47(according to the IMGT definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 48 (according to theIMGT definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 49 (according to the IMGT definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the IMGTdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67(according to the Kabat definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 52 (according to the Kabat definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53(according to the Kabat definition system), and containing no more than25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) in the framework regions as compared with the VH as setforth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively orin addition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising a CDR-L1 havingthe amino acid sequence of SEQ ID NO: 54 (according to the Kabatdefinition system), a CDR-L2 having the amino acid sequence of SEQ IDNO: 55 (according to the Kabat definition system), and a CDR-L3 havingthe amino acid sequence of SEQ ID NO: 50 (according to the Kabatdefinition system), and containing no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in theframework regions as compared with the VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 51, SEQ ID NO: 64, or SEQ ID NO: 67(according to the Kabat definition system), a CDR-H2 having the aminoacid sequence of SEQ ID NO: 52 (according to the Kabat definitionsystem), a CDR-H3 having the amino acid sequence of SEQ ID NO: 53(according to the Kabat definition system), and is at least 75% (e.g.,75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regionsto the VH as set forth in SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID NO: 68.Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody of the present disclosure comprises a humanized VL comprising aCDR-L1 having the amino acid sequence of SEQ ID NO: 54 (according to theKabat definition system), a CDR-L2 having the amino acid sequence of SEQID NO: 55 (according to the Kabat definition system), and a CDR-L3having the amino acid sequence of SEQ ID NO: 50 (according to the Kabatdefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 56 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 58 (according to the Chothia definitionsystem), and containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the frameworkregions as compared with the VH as set forth in SEQ ID NO: 61, SEQ IDNO: 65, SEQ ID NO: 68. Alternatively or in addition (e.g., in addition),the humanized anti-TfR antibody of the present disclosure comprises ahumanized VL comprising a CDR-L1 having the amino acid sequence of SEQID NO: 59 (according to the Chothia definition system), a CDR-L2 havingthe amino acid sequence of SEQ ID NO: 49 (according to the Chothiadefinition system), and a CDR-L3 having the amino acid sequence of SEQID NO: 60 (according to the Chothia definition system), and containingno more than 25 amino acid variations (e.g., no more than 25, 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 amino acid variation) in the framework regions as compared withthe VL as set forth in SEQ ID NO: 62.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising a CDR-H1 having the aminoacid sequence of SEQ ID NO: 56 (according to the Chothia definitionsystem), a CDR-H2 having the amino acid sequence of SEQ ID NO: 57(according to the Chothia definition system), a CDR-H3 having the aminoacid sequence of SEQ ID NO: 58 (according to the Chothia definitionsystem), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or99%) identical in the framework regions to the VH as set forth in SEQ IDNO: 61, SEQ ID NO: 65, SEQ ID NO: 68. Alternatively or in addition(e.g., in addition), the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VL comprising a CDR-L1 having the aminoacid sequence of SEQ ID NO: 59 (according to the Chothia definitionsystem), a CDR-L2 having the amino acid sequence of SEQ ID NO: 49(according to the Chothia definition system), and a CDR-L3 having theamino acid sequence of SEQ ID NO: 60 (according to the Chothiadefinition system), and is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%,98%, or 99%) identical in the framework regions to the VL as set forthin SEQ ID NO: 62.

Examples of amino acid sequences of the humanized anti-TfR antibodiesdescribed herein are provided in Table 3.

TABLE 3 Variable Regions of Humanized Anti-TfR Antibodies AntibodyVariable Region Amino Acid Sequence** 3A4 V_(H): VH3EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWID (N54T*)/PETGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGL Vκ4DYWGQGTLVTVSS (SEQ ID NO: 69) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3A4 V_(H): VH3EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWID (N54S*)/PESGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGL Vκ4DYWGQGTLVTVSS (SEQ ID NO: 71) V_(L):DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3A4 VH: VH3/Vκ4EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDPENGDTEYASKFQDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSS (SEQ ID NO: 72) VL:DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIK (SEQ ID NO: 70) 3M12 VH: VH3/Vκ2QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTLVTVSS (SEQ ID NO: 73) VL:DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 74)3M12 VH: VH3/Vκ3 QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTLVTVSS (SEQ ID NO: 73) VL:DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 75)3M12 VH: VH4/Vκ2 QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 76) VL:DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 74)3M12 VH: VH4/Vκ3 QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFDGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSS (SEQ ID NO: 76) VL:DIQMTQSPSSLSASVGDRVTITCRASQDISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGHTLPYTFGQGTKLEIK (SEQ ID NO: 75)5H12 VH: VH5 QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWI(C33Y*)/ YPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPY Vκ3HGMDYWGQGTLVTVSS (SEQ ID NO: 77) VL:DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 78) 5H12 VH: VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWI (C33D*)/YPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPY Vκ4HGMDYWGQGTLVTVSS (SEQ ID NO: 79) VL:DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 80) 5H12 VH: VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWI (C33Y*)/YPGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPY Vκ4HGMDYWGQGTLVTVSS (SEQ ID NO: 77) VL:DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSSEDPWTFGQGTKLEIK (SEQ ID NO: 80) *mutation positions are according to Kabatnumbering of the respective VH sequences containing the mutations **CDRsaccording to the Kabat numbering system are bolded

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising the CDR-H1, CDR-H2, andCDR-H3 of any one of the anti-TfR antibodies provided in Table 2 andcomprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)amino acid variations in the framework regions as compared with therespective humanized VH provided in Table 3. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody of thepresent disclosure comprises a humanized VL comprising the CDR-L1,CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided inTable 2 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) amino acid variations in the framework regions as compared withthe respective humanized VL provided in Table 3.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 69, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 69 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 71, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 71 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 72, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 70. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 72 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 74. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 73 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 73, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 75. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 73 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 75.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 74. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 76 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 74.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 76, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 75. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 76 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 75.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 78. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 77 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 79, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 80. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 79 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a humanized VH comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 77, and/or (e.g., and) a humanized VL comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 80. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a humanized VHcomprising the amino acid sequence of SEQ ID NO: 77 and a humanized VLcomprising the amino acid sequence of SEQ ID NO: 80.

In some embodiments, the humanized anti-TfR antibody described herein isa full-length IgG, which can include a heavy constant region and a lightconstant region from a human antibody. In some embodiments, the heavychain of any of the anti-TfR antibodies as described herein maycomprises a heavy chain constant region (CH) or a portion thereof (e.g.,CH1, CH2, CH3, or a combination thereof). The heavy chain constantregion can of any suitable origin, e.g., human, mouse, rat, or rabbit.In one specific example, the heavy chain constant region is from a humanIgG (a gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of ahuman IgG1 constant region is given below:

(SEQ ID NO: 81) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

In some embodiments, the heavy chain of any of the anti-TfR antibodiesdescribed herein comprises a mutant human IgG1 constant region. Forexample, the introduction of LALA mutations (a mutant derived from mAbb12 that has been mutated to replace the lower hinge residues Leu234Leu235 with Ala234 and Ala235) in the CH2 domain of human IgG1 is knownto reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. etal. (2000)). The mutant human IgG1 constant region is provided below(mutations bonded and underlined):

(SEQ ID NO: 82) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

In some embodiments, the light chain of any of the anti-TfR antibodiesdescribed herein may further comprise a light chain constant region(CL), which can be any CL known in the art. In some examples, the CL isa kappa light chain. In other examples, the CL is a lambda light chain.In some embodiments, the CL is a kappa light chain, the sequence ofwhich is provided below:

(SEQ ID NO: 83) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

Other antibody heavy and light chain constant regions are well known inthe art, e.g., those provided in the IMGT database (www.imgt.org) or atwww.vbase2.org/vbstat.php., both of which are incorporated by referenceherein.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising any one of the VH as listed in Table3 or any variants thereof and a heavy chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, thehumanized anti-TfR antibody described herein comprises a heavy chaincomprising any one of the VH as listed in Table 3 or any variantsthereof and a heavy chain constant region that contains no more than 25amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacid variation) as compared with SEQ ID NO: 81 or SEQ ID NO: 82. In someembodiments, the humanized anti-TfR antibody described herein comprisesa heavy chain comprising any one of the VH as listed in Table 3 or anyvariants thereof and a heavy chain constant region as set forth in SEQID NO: 81. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises heavy chain comprising any one of the VH aslisted in Table 3 or any variants thereof and a heavy chain constantregion as set forth in SEQ ID NO: 82.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a light chain comprising any one of the VL as listed in Table3 or any variants thereof and a light chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfRantibody described herein comprises a light chain comprising any one ofthe VL as listed in Table 3 or any variants thereof and a light chainconstant region contains no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withSEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises a light chain comprising any one of the VL aslisted in Table 3 or any variants thereof and a light chain constantregion set forth in SEQ ID NO: 83.

Examples of IgG heavy chain and light chain amino acid sequences of theanti-TfR antibodies described are provided in Table 4 below.

TABLE 4 Heavy chain and light chain sequences ofexamples of humanized anti-TfR IgGs AntibodyIgG Heavy Chain/Light Chain Sequences** 3A4Heavy Chain (with wild type human IgG1 constant region) VH3EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDP (N54T*)/ETGDTEYASKF Q DRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLD Vκ4YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM Q HLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3A4 Heavy Chain (with wild type human IgG1 constant region) VH3EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDP (N54S*)/ESGDTEYASKF Q DRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDY Vκ4WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM Q HLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3A4 Heavy Chain (with wild type human IgG1 constant region) VH3/EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDP Vκ4 ENGDTEYASKF QDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 87)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM Q HLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3M12 Heavy Chain (with wild type human IgG1 constant region) VH3/QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITF Vκ2DGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with wild type human IgG1 constant region) VH3/QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITF Vκ3DGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 88)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 3M12Heavy Chain (with wild type human IgG1 constant region) VH4/QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFD Vκ2GANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with wild type human IgG1 constant region) VH4/QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFD Vκ3GANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 91)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 5H12Heavy Chain (with wild type human IgG1 constant region) VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIY (C33Y*)/PGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYH Vκ3GMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)Light Chain (with kappa light chain constant region)DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQ SSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93)5H12 Heavy Chain (with wild type human IgG1 constant region) VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIY (C33D*)/PGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYH Vκ4GMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 94)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ SSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)5H12 Heavy Chain (with wild type human IgG1 constant region) VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIY (C33Y*)/PGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYH Vκ4GMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ SSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)*mutation positions are according to Kabat numbering of the respectiveVH sequences containing the mutations **CDRs according to the Kabatnumbering system are bolded; VH/VL sequences underlined

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in any one ofSEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or in addition(e.g., in addition), the humanized anti-TfR antibody of the presentdisclosure comprises a light chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the light chain as set forth in any one ofSEQ ID NOs: 85, 89, 90, 93, and 95.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to anyone of SEQ ID NOs: 84, 86, 87, 88, 91, 92, and 94. Alternatively or inaddition (e.g., in addition), the humanized anti-TfR antibody describedherein comprises a light chain comprising an amino acid sequence that isat least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical toany one of SEQ ID NOs: 85, 89, 90, 93, and 95. In some embodiments, theanti-TfR antibody described herein comprises a heavy chain comprisingthe amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91,92, and 94. Alternatively or in addition (e.g., in addition), theanti-TfR antibody described herein comprises a light chain comprisingthe amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 84, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 86, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 86 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 87, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 88, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 91, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 93. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 93.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 94, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 94 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 92, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the anti-TfR antibody is a Fab fragment, Fab′fragment, or F(ab′)₂ fragment of an intact antibody (full-lengthantibody). Antigen binding fragment of an intact antibody (full-lengthantibody) can be prepared via routine methods (e.g., recombinantly or bydigesting the heavy chain constant region of a full length IgG using anenzyme such as papain). For example, F(ab′)₂ fragments can be producedby pepsin or papain digestion of an antibody molecule, and Fab fragmentsthat can be generated by reducing the disulfide bridges of F(ab′)₂fragments. In some embodiments, a heavy chain constant region in a Fabfragment of the anti-TfR1 antibody described herein comprises the aminoacid sequence of:ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96)

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising any one of the VH as listed in Table3 or any variants thereof and a heavy chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 96. In some embodiments, the humanized anti-TfRantibody described herein comprises a heavy chain comprising any one ofthe VH as listed in Table 3 or any variants thereof and a heavy chainconstant region that contains no more than 25 amino acid variations(e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) ascompared with SEQ ID NO: 96. In some embodiments, the humanized anti-TfRantibody described herein comprises a heavy chain comprising any one ofthe VH as listed in Table 3 or any variants thereof and a heavy chainconstant region as set forth in SEQ ID NO: 96.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a light chain comprising any one of the VL as listed in Table3 or any variants thereof and a light chain constant region that is atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99%identical to SEQ ID NO: 83. In some embodiments, the humanized anti-TfRantibody described herein comprises a light chain comprising any one ofthe VL as listed in Table 3 or any variants thereof and a light chainconstant region contains no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withSEQ ID NO: 83. In some embodiments, the humanized anti-TfR antibodydescribed herein comprises a light chain comprising any one of the VL aslisted in Table 3 or any variants thereof and a light chain constantregion set forth in SEQ ID NO: 83.

Examples of Fab heavy chain and light chain amino acid sequences of theanti-TfR antibodies described are provided in Table 5 below.

TABLE 5 Heavy chain and light chain sequencesof examples of humanized anti-TfR Fabs AntibodyFab Heavy Chain/Light Chain Sequences** 3A4Heavy Chain (with partial human IgGl constant region) VH3EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDP (N54T*)/ETGDTEYASKF Q DRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLD Vκ4YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 97)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM Q HLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3A4 Heavy Chain (with partial human IgG1 constant region) VH3EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDP (N54S*)/ESGDTEYASKF Q DRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDY Vκ4WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 98)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3A4 Heavy Chain (with partial human IgG1 constant region) VH3/EVQLVQSGSELKKPGASVKVSCTASGFNIKDDYMYWVRQPPGKGLEWIGWIDP Vκ4 ENGDTEYASKF QDRVTVTADTSTNTAYMELSSLRSEDTAVYYCTLWLRRGLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 99)Light Chain (with kappa light chain constant region)DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGYTYLFWFQQRPGQSPRLLIYRMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCM Q HLEYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85)3M12 Heavy Chain (with partial human IgG1 constant region) VH3/QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITF Vκ2DGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 100)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with partial human IgG1 constant region) VH3/QVQLQESGPGLVKPSQTLSLTCSVTGYSITSGYYWNWIRQPPGKGLEWMGYITF Vκ3DGANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 100)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 3M12Heavy Chain (with partial human IgG1 constant region) VH4/QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFD Vκ2GANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 101)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 89) 3M12Heavy Chain (with partial human IgG1 constant region) VH4/QVQLQESGPGLVKPSQTLSLTCTVTGYSITSGYYWNWIRQPPGKGLEWIGYITFD Vκ3GANNYNPSLKNRVSISRDTSKNQFSLKLSSVTAEDTATYYCTRSSYDYDVLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 101)Light Chain (with kappa light chain constant region)DIQMTQSPSSLSASVGDRVTITCRAS Q DISNFLNWYQQKPGQPVKLLIYYTSRLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ GHTLPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 90) 5H12Heavy Chain (with partial human IgG1 constant region) VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIY (C33Y*)/PGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYH Vκ3GMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 102)Light Chain (with kappa light chain constant region)DIVLTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSRTDFTLTISSLQAEDVAVYYCQQ SSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 93)5H12 Heavy Chain (with partial human IgG1 constant region) VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYDINWVRQAPGQGLEWMGWIY (C33D*)/PGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYH Vκ4GMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 103)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ SSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)5H12 Heavy Chain (with partial human IgG1 constant region) VH5QVQLVQSGAEVKKPGASVKVSCKASGYSFTDYYINWVRQAPGQGLEWMGWIY (C33Y*)/PGSGNTRYSERFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCAREDYYPYH Vκ4GMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 102)Light Chain (with kappa light chain constant region)DIVMTQSPDSLAVSLGERATINCRASESVDGYDNSFMHWYQQKPGQPPKLLIFRASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQ SSEDPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 95)*mutation positions are according to Kabat numbering of the respectiveVH sequences containing the mutations **CDRs according to the Kabatnumbering system are bolded; VH/VL sequences underlined

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain containing no more than 25 amino acidvariations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acidvariation) as compared with the heavy chain as set forth in any one ofSEQ ID NOs: 97-103. Alternatively or in addition (e.g., in addition),the humanized anti-TfR antibody of the present disclosure comprises alight chain containing no more than 25 amino acid variations (e.g., nomore than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as compared withthe light chain as set forth in any one of SEQ ID NOs: 85, 89, 90, 93,and 95.

In some embodiments, the humanized anti-TfR antibody described hereincomprises a heavy chain comprising an amino acid sequence that is atleast 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to anyone of SEQ ID NOs: 97-103.

Alternatively or in addition (e.g., in addition), the humanized anti-TfRantibody described herein comprises a light chain comprising an aminoacid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%,or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, and 95. Insome embodiments, the anti-TfR antibody described herein comprises aheavy chain comprising the amino acid sequence of any one of SEQ ID NOs:97-103. Alternatively or in addition (e.g., in addition), the anti-TfRantibody described herein comprises a light chain comprising the aminoacid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, and 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 97, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 97 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 98, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 98 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 99, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 85. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 99 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 100 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 100, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 100 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 89. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 101 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 101, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 90. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 101 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 93. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 102 and a light chaincomprising the amino acid sequence of SEQ ID NO: 93.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 103, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 103 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR antibody of the presentdisclosure comprises a heavy chain comprising an amino acid sequencethat is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identicalto SEQ ID NO: 102, and/or (e.g., and) a light chain comprising an aminoacid sequence that is at least 80% identical (e.g., 80%, 85%, 90%, 95%,98%, or 99%) to SEQ ID NO: 95. In some embodiments, the humanizedanti-TfR antibody of the present disclosure comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 102 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95.

In some embodiments, the humanized anti-TfR receptor antibodiesdescribed herein can be in any antibody form, including, but not limitedto, intact (i.e., full-length) antibodies, antigen-binding fragmentsthereof (such as Fab, Fab′, F(ab′)2, Fv), single chain antibodies,bi-specific antibodies, or nanobodies. In some embodiments, humanizedthe anti-TfR antibody described herein is a scFv. In some embodiments,the humanized anti-TfR antibody described herein is a scFv-Fab (e.g.,scFv fused to a portion of a constant region). In some embodiments, theanti-TfR receptor antibody described herein is a scFv fused to aconstant region (e.g., human IgG1 constant region as set forth in SEQ IDNO: 81 or SEQ ID NO: 82, or a portion thereof such as the Fc portion) ateither the N-terminus of C-terminus.

In some embodiments, conservative mutations can be introduced intoantibody sequences (e.g., CDRs or framework sequences) at positionswhere the residues are not likely to be involved in interacting with atarget antigen (e.g., transferrin receptor), for example, as determinedbased on a crystal structure. In some embodiments, one, two or moremutations (e.g., amino acid substitutions) are introduced into the Fcregion of an anti-TfR antibody described herein (e.g., in a CH2 domain(residues 231-340 of human IgG1) and/or (e.g., and) CH3 domain (residues341-447 of human IgG1) and/or (e.g., and) the hinge region, withnumbering according to the Kabat numbering system (e.g., the EU index inKabat)) to alter one or more functional properties of the antibody, suchas serum half-life, complement fixation, Fc receptor binding and/or(e.g., and) antigen-dependent cellular cytotoxicity.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425. The number of cysteine residues in the hingeregion of the CH1 domain can be altered to, e.g., facilitate assembly ofthe light and heavy chains, or to alter (e.g., increase or decrease) thestability of the antibody or to facilitate linker conjugation.

In some embodiments, one, two or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of a muscle-targetingantibody described herein (e.g., in a CH2 domain (residues 231-340 ofhuman IgG1) and/or (e.g., and) CH3 domain (residues 341-447 of humanIgG1) and/or (e.g., and) the hinge region, with numbering according tothe Kabat numbering system (e.g., the EU index in Kabat)) to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, which are incorporatedherein by reference.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo.

In some embodiments, one, two or more amino acid mutations (i.e.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to decrease the half-life of the anti-anti-TfRantibody in vivo. In some embodiments, one, two or more amino acidmutations (i.e., substitutions, insertions or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In some embodiments, the antibodies can haveone or more amino acid mutations (e.g., substitutions) in the secondconstant (CH2) domain (residues 231-340 of human IgG1) and/or (e.g.,and) the third constant (CH3) domain (residues 341-447 of human IgG1),with numbering according to the EU index in Kabat (Kabat E A et al.,(1991) supra). In some embodiments, the constant region of the IgG1 ofan antibody described herein comprises a methionine (M) to tyrosine (Y)substitution in position 252, a serine (S) to threonine (T) substitutionin position 254, and a threonine (T) to glutamic acid (E) substitutionin position 256, numbered according to the EU index as in Kabat. SeeU.S. Pat. No. 7,658,921, which is incorporated herein by reference. Thistype of mutant IgG, referred to as “YTE mutant” has been shown todisplay fourfold increased half-life as compared to wild-type versionsof the same antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281:23514-24). In some embodiments, an antibody comprises an IgG constantdomain comprising one, two, three or more amino acid substitutions ofamino acid residues at positions 251-257, 285-290, 308-314, 385-389, and428-436, numbered according to the EU index as in Kabat.

In some embodiments, one, two or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the anti-anti-TfR antibody. The effector ligand to whichaffinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, thedeletion or inactivation (through point mutations or other means) of aconstant region domain can reduce Fc receptor binding of the circulatingantibody thereby increasing tumor localization. See, e.g., U.S. Pat.Nos. 5,585,097 and 8,591,886 for a description of mutations that deleteor inactivate the constant domain and thereby increase tumorlocalization. In some embodiments, one or more amino acid substitutionsmay be introduced into the Fc region of an antibody described herein toremove potential glycosylation sites on Fc region, which may reduce Fcreceptor binding (see, e.g., Shields R L et al., (2001) J Biol Chem 276:6591-604).

In some embodiments, one or more amino in the constant region of ananti-TfR antibody described herein can be replaced with a differentamino acid residue such that the antibody has altered C1q binding and/or(e.g., and) reduced or abolished complement dependent cytotoxicity(CDC). This approach is described in further detail in U.S. Pat. No.6,194,551 (Idusogie et al). In some embodiments, one or more amino acidresidues in the N-terminal region of the CH2 domain of an antibodydescribed herein are altered to thereby alter the ability of theantibody to fix complement. This approach is described further inInternational Publication No. WO 94/29351. In some embodiments, the Fcregion of an antibody described herein is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or (e.g., and) to increase the affinity of theantibody for an Fcγ receptor. This approach is described further inInternational Publication No. WO 00/42072.

In some embodiments, the heavy and/or (e.g., and) light chain variabledomain(s) sequence(s) of the antibodies provided herein can be used togenerate, for example, CDR-grafted, chimeric, humanized, or compositehuman antibodies or antigen-binding fragments, as described elsewhereherein. As understood by one of ordinary skill in the art, any variant,CDR-grafted, chimeric, humanized, or composite antibodies derived fromany of the antibodies provided herein may be useful in the compositionsand methods described herein and will maintain the ability tospecifically bind transferrin receptor, such that the variant,CDR-grafted, chimeric, humanized, or composite antibody has at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95% or more binding to transferrin receptor relative to the originalantibody from which it is derived.

In some embodiments, the antibodies provided herein comprise mutationsthat confer desirable properties to the antibodies. For example, toavoid potential complications due to Fab-arm exchange, which is known tooccur with native IgG4 mAbs, the antibodies provided herein may comprisea stabilizing ‘Adair’ mutation (Angal S., et al., “A single amino acidsubstitution abolishes the heterogeneity of chimeric mouse/human (IgG4)antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EUnumbering; residue 241 Kabat numbering) is converted to prolineresulting in an IgG1-like hinge sequence. Accordingly, any of theantibodies may include a stabilizing ‘Adair’ mutation.

In some embodiments, an antibody is modified, e.g., modified viaglycosylation, phosphorylation, sumoylation, and/or (e.g., and)methylation. In some embodiments, an antibody is a glycosylatedantibody, which is conjugated to one or more sugar or carbohydratemolecules. In some embodiments, the one or more sugar or carbohydratemolecule are conjugated to the antibody via N-glycosylation,O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment),and/or (e.g., and) phosphoglycosylation. In some embodiments, the one ormore sugar or carbohydrate molecules are monosaccharides, disaccharides,oligosaccharides, or glycans. In some embodiments, the one or more sugaror carbohydrate molecule is a branched oligosaccharide or a branchedglycan. In some embodiments, the one or more sugar or carbohydratemolecule includes a mannose unit, a glucose unit, an N-acetylglucosamineunit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, ora phospholipid unit. In some embodiments, there are about 1-10, about1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. Insome embodiments, a glycosylated antibody is fully or partiallyglycosylated. In some embodiments, an antibody is glycosylated bychemical reactions or by enzymatic means. In some embodiments, anantibody is glycosylated in vitro or inside a cell, which may optionallybe deficient in an enzyme in the N- or O-glycosylation pathway, e.g. aglycosyltransferase. In some embodiments, an antibody is functionalizedwith sugar or carbohydrate molecules as described in InternationalPatent Application Publication WO2014065661, published on May 1, 2014,entitled, “Modified antibody, antibody-conjugate and process for thepreparation thereof”.

In some embodiments, any one of the anti-TfR1 antibodies describedherein may comprise a signal peptide in the heavy and/or (e.g., and)light chain sequence (e.g., a N-terminal signal peptide). In someembodiments, the anti-TfR1 antibody described herein comprises any oneof the VH and VL sequences, any one of the IgG heavy chain and lightchain sequences, or any one of the Fab heavy chain and light chainsequences described herein, and further comprises a signal peptide(e.g., a N-terminal signal peptide). In some embodiments, the signalpeptide comprises the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ IDNO: 104).

III Preparation of the Anti-TfR Antibodies

Antibodies capable of binding TfR as described herein can be made by anymethod known in the art. See, for example, Harlow and Lane, (1998)Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NewYork.

In some embodiments, antibodies specific to a target antigen (e.g., TfR)can be made by the conventional hybridoma technology. The full-lengthtarget antigen or a fragment thereof, optionally coupled to a carrierprotein such as KLH, can be used to immunize a host animal forgenerating antibodies binding to that antigen. The route and schedule ofimmunization of the host animal are generally in keeping withestablished and conventional techniques for antibody stimulation andproduction, as further described herein. General techniques forproduction of mouse, humanized, and human antibodies are known in theart and are described herein. It is contemplated that any mammaliansubject including humans or antibody producing cells therefrom can bemanipulated to serve as the basis for production of mammalian, includinghuman hybridoma cell lines. Typically, the host animal is inoculatedintraperitoneally, intramuscularly, orally, subcutaneously,intraplantar, and/or (e.g., and) intradermally with an amount ofimmunogen, including as described herein.

If desired, an antibody (monoclonal or polyclonal) of interest (e.g.,produced by a hybridoma) may be sequenced and the polynucleotidesequence may then be cloned into a vector for expression or propagation.The sequence encoding the antibody of interest may be maintained invector in a host cell and the host cell can then be expanded and frozenfor future use. In an alternative, the polynucleotide sequence may beused for genetic manipulation to “humanize” the antibody or to improvethe affinity (affinity maturation), or other characteristics of theantibody. For example, the constant region may be engineered to moreresemble human constant regions to avoid immune response if the antibodyis used in clinical trials and treatments in humans. It may be desirableto genetically manipulate the antibody sequence to obtain greateraffinity to the target antigen and greater efficacy. It will be apparentto one of skill in the art that one or more polynucleotide changes canbe made to the antibody and still maintain its binding specificity tothe target antigen.

In other embodiments, fully human antibodies can be obtained by usingcommercially available mice that have been engineered to expressspecific human immunoglobulin proteins. Transgenic animals that aredesigned to produce a more desirable (e.g., fully human antibodies) ormore robust immune response may also be used for generation of humanizedor human antibodies. Examples of such technology are XenomouseR™ fromAmgen, Inc. (Fremont, Calif.) and HuMAb-MouseR™ and TC Mouse™ fromMedarex, Inc. (Princeton, N.J.) or H2L2 mice from Harbour Antibodies BV(Holland). In another alternative, antibodies may be made recombinantlyby phage display or yeast technology. See, for example, U.S. Pat. Nos.5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al.,(1994) Annu. Rev. Immunol. 12:433-455. Alternatively, the phage displaytechnology (McCafferty et al., (1990) Nature 348:552-553) can be used toproduce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fab′fragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments. Genetically engineered antibodies, such as humanizedantibodies, chimeric antibodies, single-chain antibodies, andbi-specific antibodies, can be produced via, e.g., conventionalrecombinant technology. In one example, DNA encoding a monoclonalantibodies specific to a target antigen can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAmay be placed into one or more expression vectors, which are thentransfected into host cells such as E. coli cells, simian COS cells,Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. See,e.g., PCT Publication No. WO 87/04462. The DNA can then be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide. In that manner,genetically engineered antibodies, such as “chimeric” or “hybrid”antibodies; can be prepared that have the binding specificity of atarget antigen.

A single-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region.Preferably, a flexible linker is incorporated between the two variableregions.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted toproduce a phage or yeast scFv library and scFv clones specific to TfRcan be identified from the library following routine procedures.Positive clones can be subjected to further screening to identify thosethat has high TfR binding affinity.

Antibodies obtained following a method known in the art and describedherein can be characterized using methods well known in the art. Forexample, one method is to identify the epitope to which the antigenbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In one example,epitope mapping can be accomplished use H/D-Ex (hydrogen deuteriumexchange) coupled with proteolysis and mass spectrometry. In anadditional example, epitope mapping can be used to determine thesequence to which an antibody binds. The epitope can be a linearepitope, i.e., contained in a single stretch of amino acids, or aconformational epitope formed by a three-dimensional interaction ofamino acids that may not necessarily be contained in a single stretch(primary structure linear sequence). Peptides of varying lengths (e.g.,at least 4-6 amino acids long) can be isolated or synthesized (e.g.,recombinantly) and used for binding assays with an antibody. In anotherexample, the epitope to which the antibody binds can be determined in asystematic screening by using overlapping peptides derived from thetarget antigen sequence and determining binding by the antibody.According to the gene fragment expression assays, the open reading frameencoding the target antigen is fragmented either randomly or by specificgenetic constructions and the reactivity of the expressed fragments ofthe antigen with the antibody to be tested is determined. The genefragments may, for example, be produced by PCR and then transcribed andtranslated into protein in vitro, in the presence of radioactive aminoacids. The binding of the antibody to the radioactively labeled antigenfragments is then determined by immunoprecipitation and gelelectrophoresis. Certain epitopes can also be identified by using largelibraries of random peptide sequences displayed on the surface of phageparticles (phage libraries). Alternatively, a defined library ofoverlapping peptide fragments can be tested for binding to the testantibody in simple binding assays. In an additional example, mutagenesisof an antigen binding domain, domain swapping experiments and alaninescanning mutagenesis can be performed to identify residues required,sufficient, and/or (e.g., and) necessary for epitope binding.Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope as the other antibodies. Competitionassays are well known to those of skill in the art.

In some examples, an anti-TfR antibody is prepared by recombinanttechnology as exemplified below. Nucleic acids encoding the heavy andlight chain of an anti-TfR antibody as described herein can be clonedinto one expression vector, each nucleotide sequence being in operablelinkage to a suitable promoter. In one example, each of the nucleotidesequences encoding the heavy chain and light chain is in operablelinkage to a distinct promoter. Alternatively, the nucleotide sequencesencoding the heavy chain and the light chain can be in operable linkagewith a single promoter, such that both heavy and light chains areexpressed from the same promoter. When necessary, an internal ribosomalentry site (IRES) can be inserted between the heavy chain and lightchain encoding sequences.

In some examples, the nucleotide sequences encoding the two chains ofthe antibody are cloned into two vectors, which can be introduced intothe same or different cells. When the two chains are expressed indifferent cells, each of them can be isolated from the host cellsexpressing such and the isolated heavy chains and light chains can bemixed and incubated under suitable conditions allowing for the formationof the antibody.

Generally, a nucleic acid sequence encoding one or all chains of anantibody can be cloned into a suitable expression vector in operablelinkage with a suitable promoter using methods known in the art. Forexample, the nucleotide sequence and vector can be contacted, undersuitable conditions, with a restriction enzyme to create complementaryends on each molecule that can pair with each other and be joinedtogether with a ligase. Alternatively, synthetic nucleic acid linkerscan be ligated to the termini of a gene. These synthetic linkers containnucleic acid sequences that correspond to a particular restriction sitein the vector. The selection of expression vectors/promoter would dependon the type of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the antibodiesdescribed herein, including, but not limited to, cytomegalovirus (CMV)intermediate early promoter, a viral LTR such as the Rous sarcoma virusLTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E.coli lac UV promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promotersinclude those using the lac repressor from E. coli as a transcriptionmodulator to regulate transcription from lac operator bearing mammaliancell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those usingthe tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc.Natl. Acad. Sci. USA 89:5547-555115 (1992); Yao, F. et al., Human GeneTherapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad.Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16or p65 using astradiol, RU486, diphenol murislerone, or rapamycin.Inducible systems are available from Invitrogen, Clontech and Ariad,among others.

Regulatable promoters that include a repressor with the operon can beused. In one embodiment, the lac repressor from E. coli can function asa transcriptional modulator to regulate transcription from lacoperator-bearing mammalian cell promoters [M. Brown et al., Cell,49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl.Acad. Sci. USA, 89:5547-5551(1992)] combined the tetracycline repressor(tetR) with the transcription activator (VP 16) to create atetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP16), with the tetO bearing minimal promoter derived from the humancytomegalovirus (hCMV) promoter to create a tetR-tet operator system tocontrol gene expression in mammalian cells. In one embodiment, atetracycline inducible switch is used. The tetracycline repressor (tetR)alone, rather than the tetR-mammalian cell transcription factor fusionderivatives can function as potent trans-modulator to regulate geneexpression in mammalian cells when the tetracycline operator is properlypositioned downstream for the TATA element of the CMVIE promoter (Yao etal., Human Gene Therapy). One particular advantage of this tetracyclineinducible switch is that it does not require the use of a tetracyclinerepressor-mammalian cells transactivator or repressor fusion protein,which in some instances can be toxic to cells (Gossen et al., Natl.Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad.Sci. USA, 92:6522-6526 (1995)), to achieve its regulatable effects.

Additionally, the vector can contain, for example, some or all of thefollowing: a selectable marker gene, such as the neomycin gene forselection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; SV40 polyoma origins ofreplication and ColE1 for proper episomal replication; internal ribosomebinding sites (IRESes), versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Suitable vectors and methods for producing vectors containing transgenesare well known and available in the art. Examples of polyadenylationsignals useful to practice the methods described herein include, but arenot limited to, human collagen I polyadenylation signal, human collagenII polyadenylation signal, and SV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) comprising nucleic acidsencoding any of the antibodies may be introduced into suitable hostcells for producing the antibodies. The host cells can be cultured undersuitable conditions for expression of the antibody or any polypeptidechain thereof. Such antibodies or polypeptide chains thereof can berecovered by the cultured cells (e.g., from the cells or the culturesupernatant) via a conventional method, e.g., affinity purification. Ifnecessary, polypeptide chains of the antibody can be incubated undersuitable conditions for a suitable period of time allowing forproduction of the antibody.

In some embodiments, methods for preparing an antibody described hereininvolve a recombinant expression vector that encodes both the heavychain and the light chain of an anti-TfR antibody, as also describedherein. The recombinant expression vector can be introduced into asuitable host cell (e.g., a dhfr-CHO cell) by a conventional method,e.g., calcium phosphate mediated transfection. Positive transformanthost cells can be selected and cultured under suitable conditionsallowing for the expression of the two polypeptide chains that form theantibody, which can be recovered from the cells or from the culturemedium. When necessary, the two chains recovered from the host cells canbe incubated under suitable conditions allowing for the formation of theantibody. In some embodiments, the host cell used for expressing theanti-TfR antibodies described herein are CHO-S cells (e.g., ThermoFisherCatalog #R80007).

In one example, two recombinant expression vectors are provided, oneencoding the heavy chain of the anti-TfR antibody and the other encodingthe light chain of the anti-TfR antibody. Both of the two recombinantexpression vectors can be introduced into a suitable host cell (e.g.,dhfr-CHO cell) by a conventional method, e.g., calciumphosphate-mediated transfection.

Alternatively, each of the expression vectors can be introduced into asuitable host cells. Positive transformants can be selected and culturedunder suitable conditions allowing for the expression of the polypeptidechains of the antibody. When the two expression vectors are introducedinto the same host cells, the antibody produced therein can be recoveredfrom the host cells or from the culture medium. If necessary, thepolypeptide chains can be recovered from the host cells or from theculture medium and then incubated under suitable conditions allowing forformation of the antibody. When the two expression vectors areintroduced into different host cells, each of them can be recovered fromthe corresponding host cells or from the corresponding culture media.The two polypeptide chains can then be incubated under suitableconditions for formation of the antibody.

Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recovery of the antibodiesfrom the culture medium. For example, some antibodies can be isolated byaffinity chromatography with a Protein A or Protein G coupled matrix.

In some embodiments, any one of the anti-TfR antibodies described hereinis produced by recombinant DNA technology in Chinese hamster ovary (CHO)cell suspension culture, optionally in CHO-Kl cell (e.g., CHO-Kl cellsderived from European Collection of Animal Cell Culture, Cat. No.85051005) suspension culture.

In some embodiments, an antibody provided herein may have one or morepost-translational modifications. In some embodiments, N-terminalcyclization, also called pyroglutamate formation (pyro-Glu), may occurin the antibody at N-terminal Glutamate (Glu) and/or Glutamine (Gln)residues during production. As such, it should be appreciated that anantibody specified as having a sequence comprising an N-terminalglutamate or glutamine residue encompasses antibodies that haveundergone pyroglutamate formation resulting from a post-translationalmodification. In some embodiments, pyroglutamate formation occurs in aheavy chain sequence. In some embodiments, pyroglutamate formationoccurs in a light chain sequence.

IV. Complexes

In some embodiments, the humanized anti-TfR antibodies described hereincan be used for delivering a molecular payload to a target cell or atarget tissue (e.g., a cell or tissue that expresses TfR). Accordingly,some aspects of the present disclosure provide complexes comprising anyone of the humanized anti-TfR antibody described herein (e.g., humanized3-A4, 3-M12, or 5-H12 in IgG or Fab form as provided in Table 4 andTable 5) to a molecular payload. The complexes described herein may beused in various applications, e.g., diagnostic or therapeuticapplications.

In some embodiments, a complex comprises an anti-TfR antibody covalentlylinked to an oligonucleotide (e.g., an antisense oligonucleotide). Insome embodiments, the complex described herein is used to modulate theactivity or function of at least one gene, protein, and/or (e.g., and)nucleic acid. In some embodiments, the molecular payload present with acomplex is responsible for the modulation of a gene, protein, and/or(e.g., and) nucleic acids. A molecular payload may be a small molecule,protein, nucleic acid, oligonucleotide, or any molecular entity capableof modulating the activity or function of a gene, protein, and/or (e.g.,and) nucleic acid in a cell. In some embodiments, a molecular payload isan oligonucleotide that targets a disease-associated repeat in musclecells.

A. Molecular Payloads

Some aspects of the disclosure provide molecular payloads, e.g., formodulating a biological outcome, e.g., the transcription of a DNAsequence, the expression of a protein, or the activity of a protein,that can be linked to any one of the anti-TfR antibodies describedherein. In some embodiments, such molecular payloads are capable oftargeting to a muscle cell, e.g., via specifically binding to a nucleicacid or protein in the muscle cell following delivery to the muscle cellby the linked anti-TfR antibody. It should be appreciated that varioustypes of molecular payloads may be used in accordance with thedisclosure. For example, the molecular payload may comprise, or consistof, an oligonucleotide (e.g., antisense oligonucleotide), a peptide(e.g., a peptide that binds a nucleic acid or protein associated withdisease in a muscle cell), a protein (e.g., a protein that binds anucleic acid or protein associated with disease in a muscle cell), or asmall molecule (e.g., a small molecule that modulates the function of anucleic acid or protein associated with disease in a muscle cell).

In some embodiments, the molecular payload is an oligonucleotide thatcomprises a strand having a region of complementarity to a gene providedin Table 6.

TABLE 6 List of muscle diseases and corresponding genes. Rare MuscleDisease Target Genes Disease Gene Symbol GenBank Accession No. AdultPompe GAA NM_000152; NM_001079803; NM_001079804 Adult Pompe GYS1NM_001161587; NM_002103 Centronuclear myopathy (CNM) DNM2 NM_001190716;NM_004945; NM_001005362; NM_001005360; NM_001005361; NM_007871 Duchennemuscular dystrophy DMD NM_004023; NM_004020; NM_004018; NM_004012Facioscapulohumeral muscular DUX4 NM_001306068; dystrophy (FSHD)NM_001363820; NM_001205218; NM_001293798 Familial hypertrophic MYBPC3NM_000256 cardiomyopathy Familial hypertrophic MYH6 NM_002471;NM_001164171; cardiomyopathy NM_010856 Familial hypertrophic MYH7NM_000257; NM_080728 cardiomyopathy Familial hypertrophic TNNI3NM_000363 cardiomyopathy Familial hypertrophic TNNT2 NM_001001432;cardiomyopathy NM_001001431; NM_000364; NM_001001430; NM_001276347;NM_001276346; NM_001276345 Fibrodysplasia Ossificans ACVR1 NM_001105;NM_001347663; Progressiva (FOP) NM_001347664; NM_001347665;NM_001347666; NM_001347667; NM_001111067 Friedreich’s ataxia (FRDA) FXNNM_001161706; NM_181425; NM_000144 Inclusion body myopathy 2 GNENM_001190383; NM_001190384; NM_001128227; NM_005476; NM_001190388 Laingdistal myopathy MYH7 NM_000257; NM_080728 Myofibrillar myopathy BAG3NM_004281 Myofibrillar myopathy CRYAB NM_001885; NM_001330379;NM_001289807; NM_001289808 Myofibrillar myopathy DES NM_001927Myofibrillar myopathy DNAJB6 NM_005494; NM_058246 Myofibrillar myopathyFHL1 NM_001159701; NM_001159699; NM_001159702; NM_001159703;NM_001159704; NM_001159700; NM_001167819; NM_001330659; NM_001449;NM_001077362 Myofibrillar myopathy FLNC NM_001458; NM_001127487Myofibrillar myopathy LDB3 NM_007078; NM_001171611; NM_001171610;NM_001080114; NM_001080115; NM_001080116 Myofibrillar myopathy MYOTNM_001300911; NM_006790; NM_001135940 Myofibrillar myopathy PLECNM_201378; NM_201379; NM_201380; NM_201381; NM_201382; NM_201383;NM_201384; NM_000445 Myofibrillar myopathy TTN NM_133432; NM_133379;NM_133437; NM_003319; NM_001256850; NM_001267550; NM_133378 Myotoniacongenita (autosomal CLCN1 NM_000083; NM_013491 dominant form, ThomsenDisease) Myotonic dystrophy type I DMPK NM_001081563; NM_004409;NM_001081560; NM_001081562; NM_001288764; NM_001288765; NM_001288766Myotonic dystrophy type II CNBP NM_001127192; NM_001127193;NM_001127194; NM_001127195; NM_001127196; NM_003418 Myotubular myopathyMTM1 NM_000252 Oculopharyngeal muscular dystrophy PABPN1 NM_004643Paramyotonia congenita SCN4A NM_000334 Muscle Atrophy Gene TargetsGenBank Gene Symbol Accession No. Related Publications* INHBA (alsoknown as EDF; FRP) NM_002192; Lee SJ, et al., Regulation ofXM_017012175.1; muscle mass by follistatin and XM_017012176.1;activins., Mol Endocrinol. 2010 XM_017012174.1 Oct; 24(10): 1998-2008.doi: 10.1210/me.2010-0127. Epub 2010 Sep 1. FBXO32 (also known as Fbx32;NM_058229.3; Bodine, S. C., et al., Identification MAFbx)NM_001242463.1; of ubiquitin ligases required for NM_148177.2 skeletalmuscle atrophy. Science 294: 1704-1708, 2001. Gomes, M. D., et al.,Atrogin-1, a muscle-specific F-box protein highly expressed duringmuscle atrophy. Proc. Nat. Acad. Sci. 98: 14440-14445, 2001. MSTN (alsoknown as GDF8; NM_005259.2 Saunders, M. A., et al., Human MSLHP)adaptive evolution of myostatin (GDF8), a regulator of muscle growth.Am. J. Hum. Genet. 79: 1089-1097, 2006. Lin, J., et al., Myostatinknockout in mice increases myogenesis and decreases adipogenesis.Biochem. Biophys. Res. Commun. 291: 701-706, 2002. Wei Y, et al.,Prevention of Muscle Wasting by CRISPR/Cas9-mediated Disruption ofMyostatin In Vivo Volume 24, Issue 11, p1889- 1891, November 2016 TRIM63NM_032588.3; Höllriegel R, et al. Anabolic (also known as IRF; SMRZ;XM_017002559.2 effects of exercise training in MURF1; MURF2; RNF28)patients with advanced chronic heart failure (NYHA IIIb): impact onubiquitin-protein ligases expression and skeletal muscle size. Int JCardiol, 2013 Aug. 10. Eddins MJ, et al. Targeting the ubiquitin E3ligase MuRFl to inhibit muscle atrophy. Cell Biochem Biophys, 2011 Jun.*The contents of the cited references are incorporated herein byreference in their entireties.

In some embodiments, the molecular payload is an agent for the treatmentof a neurological disorder. A “neurological disorder” as used hereinrefers to a disease or disorder which affects the CNS and/or (e.g., and)which has an etiology in the CNS. Examples of CNS diseases or disordersinclude, but are not limited to, neuropathy, amyloidosis, cancer, anocular disease or disorder, viral or microbial infection, inflammation,ischemia, neurodegenerative disease, seizure, behavioral disorders, anda lysosomal storage disease. For the purposes of this application, theCNS will be understood to include the eye, which is normally sequesteredfrom the rest of the body by the blood-retina barrier. Specific examplesof neurological disorders include, but are not limited to,neurodegenerative diseases (including, but not limited to, Lewy bodydisease, postpoliomyelitis syndrome, Shy-Draeger syndrome,olivopontocerebellar atrophy, Parkinson's disease, multiple systematrophy, striatonigral degeneration, tauopathies (including, but notlimited to, Alzheimer disease and supranuclear palsy), prion diseases(including, but not limited to, bovine spongiform encephalopathy,scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), bulbar palsy, motor neuron disease, andnervous system heterodegenerative disorders (including, but not limitedto, Canavan disease, Huntington's disease, neuronalceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkeskinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome,lafora disease, Rett syndrome, hepatolenticular degeneration,Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia(including, but not limited to, Pick's disease, and spinocerebellarataxia), cancer (e.g. of the CNS, including brain metastases resultingfrom cancer elsewhere in the body). Non-limiting, examples ofneurological disorder drugs that may be conjugated to any one of theanti-TfR antibodies described herein and the corresponding conditionsthey may treat are provided in Table 7.

TABLE 7 Examples of neurological disorder drugs and conditions treatedDrug Neurological disorder Anti-BACE1 Antibody Alzheimer's, acute andchronic brain injury, stroke Anti-Abeta Antibody Alzheimer's diseaseAnti-Tan Antibody Alzheimer's disease, taupathies Neurotrophin Stroke,acute brain injury, spinal cord injury Brain-derived neurotrophic factor(BDNF), Chronic brain injury (Neurogenesis) Fibroblast growth factor 2(FGF-2) Anti-Epidermal Growth Factor Receptor Brain Cancer(EGFR)-antibody Glial cell-line derived neural factor (GDNF) Parkinson'sdisease Brain derived neurotrophic factor (BDNF) Amyotrophic lateralsclerosis, depression Lysosomal enzyme Lysosomal storage disorders ofthe brain Ciliary neurotrophic factor (CNTF) Amyotrophic lateralsclerosis Neuregulin-1 Schizophrenia Anti-HER2 antibody (e.g.trastuzamab, Brain metastasis from HER2-positive cancer pertuzumab,etc.) Anti-BEGF antibody (e.g. bevacizumab) Recurrent or newly diagnosedglioblastoma, recurrent malignant glioma, brain metastasis

In some embodiments, at least one (e.g., at least 2, at least 3, atleast 4, at least 5, at least 10) molecular payload (e.g.oligonucleotides) is linked to any one of the anti-TfR antibodydescribed herein. In some embodiments, all molecular payloads attachedto the anti-TfR antibody are the same, e.g. target the same gene. Insome embodiments, all molecular payloads attached to the anti-TfRantibody are different, for example the molecular payloads may targetdifferent portions of the same target gene, or the molecular payloadsmay target at least two different target genes. In some embodiments, ananti-TfR antibody described herein may be attached to some molecularpayloads that are the same and some molecular payloads that aredifferent.

The present disclosure also provides a composition comprising aplurality of complexes, for which at least 80% (e.g., at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%) ofthe complexes comprise an anti-TfR antibody linked to the same number ofmolecular payloads (e.g., oligonucleotides).

Exemplary molecular payloads are described in further detail herein,however, it should be appreciated that the exemplary molecular payloadsprovided herein are not meant to be limiting.

i. Oligonucleotides

Any suitable oligonucleotide may be used as a molecular payload, asdescribed herein. In some embodiments, the oligonucleotide may bedesigned to cause degradation of an mRNA (e.g., the oligonucleotide maybe a gapmer, an siRNA, a ribozyme or an aptamer that causesdegradation). In some embodiments, the oligonucleotide may be designedto block translation of an mRNA (e.g., the oligonucleotide may be amixmer, an siRNA or an aptamer that blocks translation). In someembodiments, an oligonucleotide may be designed to caused degradationand block translation of an mRNA. In some embodiments, anoligonucleotide may be a guide nucleic acid (e.g., guide RNA) fordirecting activity of an enzyme (e.g., a gene editing enzyme). Otherexamples of oligonucleotides are provided herein. It should beappreciated that, in some embodiments, oligonucleotides in one format(e.g., antisense oligonucleotides) may be suitably adapted to anotherformat (e.g., siRNA oligonucleotides) by incorporating functionalsequences (e.g., antisense strand sequences) from one format to theother format.

In some embodiments, an oligonucleotide may comprise a region ofcomplementarity to a target gene provided in Table 6.

In some embodiments, the oligonucleotide may target lncRNA or mRNA,e.g., for degradation. In some embodiments, the oligonucleotide maytarget, e.g., for degradation, a nucleic acid encoding a proteininvolved in a mismatch repair pathway, e.g., MSH2, MutLalpha, MutSbeta,MutLalpha. Non-limiting examples of proteins involved in mismatch repairpathways, for which mRNAs encoding such proteins may be targeted byoligonucleotides described herein, are described in Iyer, R. R. et al.,“DNA triplet repeat expansion and mismatch repair” Annu Rev Biochem.2015; 84:199-226; and Schmidt M. H. and Pearson C. E.,“Disease-associated repeat instability and mismatch repair” DNA Repair(Amst). 2016 February; 38:117-26.

In some embodiments, any one of the oligonucleotides can be in saltform, e.g., as sodium, potassium, or magnesium salts.

In some embodiments, the 5′ or 3′ nucleoside (e.g., terminal nucleoside)of any one of the oligonucleotides described herein is conjugated to anamine group, optionally via a spacer. In some embodiments, the spacercomprises an aliphatic moiety. In some embodiments, the spacer comprisesa polyethylene glycol moiety. In some embodiments, a phosphodiesterlinkage is present between the spacer and the 5′ or 3′ nucleoside of theoligonucleotide. In some embodiments, the 5′ or 3′ nucleoside (e.g.,terminal nucleoside) of any of the oligonucleotides described herein isconjugated to a spacer that is a substituted or unsubstituted aliphatic,substituted or unsubstituted heteroaliphatic, substituted orunsubstituted carbocyclylene, substituted or unsubstitutedheterocyclylene, substituted or unsubstituted arylene, substituted orunsubstituted heteroarylene, —O—, —N(R^(A))—, —S—, —C(═O)—, —C(═O)O—,—C(═O)NR^(A)—, —NR^(A)C(═O)—, —NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—,—NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—, —OC(═O)—, —OC(═O)O—,—OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, or a combination thereofeach R^(A) is independently hydrogen or substituted or unsubstitutedalkyl. In certain embodiments, the spacer is a substituted orunsubstituted alkylene, substituted or unsubstituted heterocyclylene,substituted or unsubstituted heteroarylene, —O—, —N(R^(A))—, or—C(═O)N(R^(A))₂, or a combination thereof.

In some embodiments, the 5′ or 3′ nucleoside of any one of theoligonucleotides described herein is conjugated to a compound of theformula —NH₂—(CH₂)_(n)—, wherein n is an integer from 1 to 12. In someembodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, aphosphodiester linkage is present between the compound of the formulaNH₂—(CH₂)_(n)— and the 5′ or 3′ nucleoside of the oligonucleotide. Insome embodiments, a compound of the formula NH₂—(CH₂)₆— is conjugated tothe oligonucleotide via a reaction between 6-amino-1-hexanol(NH₂—(CH₂)₆—OH) and the 5′ phosphate of the oligonucleotide.

In some embodiments, the oligonucleotide is conjugated to a targetingagent, e.g., a muscle targeting agent such as an anti-TfR antibody,e.g., via the amine group.

a. Oligonucleotide Size/Sequence

Oligonucleotides may be of a variety of different lengths, e.g.,depending on the format. In some embodiments, an oligonucleotide is 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, the oligonucleotide is 8 to 50 nucleotides inlength, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 21 to 23 nucleotides in lengths, etc.

In some embodiments, a complementary nucleic acid sequence of anoligonucleotide for purposes of the present disclosure is specificallyhybridizable or specific for the target nucleic acid when binding of thesequence to the target molecule (e.g., mRNA) interferes with the normalfunction of the target (e.g., mRNA) to cause a loss of activity (e.g.,inhibiting translation) or expression (e.g., degrading a target mRNA)and there is a sufficient degree of complementarity to avoidnon-specific binding of the sequence to non-target sequences underconditions in which avoidance of non-specific binding is desired, e.g.,under physiological conditions in the case of in vivo assays ortherapeutic treatment, and in the case of in vitro assays, underconditions in which the assays are performed under suitable conditionsof stringency. Thus, in some embodiments, an oligonucleotide may be atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% or 100% complementary to the consecutivenucleotides of a target nucleic acid. In some embodiments acomplementary nucleotide sequence need not be 100% complementary to thatof its target to be specifically hybridizable or specific for a targetnucleic acid.

In some embodiments, an oligonucleotide comprises region ofcomplementarity to a target nucleic acid that is in the range of 8 to15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides inlength. In some embodiments, a region of complementarity of anoligonucleotide to a target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 nucleotides in length. In some embodiments, the region ofcomplementarity is complementary with at least 8 consecutive nucleotidesof a target nucleic acid. In some embodiments, an oligonucleotide maycontain 1, 2 or 3 base mismatches compared to the portion of theconsecutive nucleotides of target nucleic acid. In some embodiments theoligonucleotide may have up to 3 mismatches over 15 bases, or up to 2mismatches over 10 bases.

In some embodiments, the oligonucleotide is complementary (e.g., atleast 85% at least 90%, at least 95%, or 100%) to a target sequence ofany one of the oligonucleotides provided herein. In some embodiments,such target sequence is 100% complementary to the oligonucleotideprovided herein.

In some embodiments, any one or more of the thymine bases (T's) in anyone of the oligonucleotides provided herein may optionally be uracilbases (U's), and/or any one or more of the U's may optionally be T's.

b. Oligonucleotide Modifications:

The oligonucleotides described herein may be modified, e.g., comprise amodified sugar moiety, a modified internucleoside linkage, a modifiednucleotide and/or (e.g., and) combinations thereof. In addition, in someembodiments, oligonucleotides may exhibit one or more of the followingproperties: do not mediate alternative splicing; are not immunestimulatory; are nuclease resistant; have improved cell uptake comparedto unmodified oligonucleotides; are not toxic to cells or mammals; haveimproved endosomal exit internally in a cell; minimizes TLR stimulation;or avoid pattern recognition receptors. Any of the modified chemistriesor formats of oligonucleotides described herein can be combined witheach other. For example, one, two, three, four, five, or more differenttypes of modifications can be included within the same oligonucleotide.

In some embodiments, certain nucleotide modifications may be used thatmake an oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide oroligoribonucleotide molecules; these modified oligonucleotides surviveintact for a longer time than unmodified oligonucleotides. Specificexamples of modified oligonucleotides include those comprising modifiedbackbones, for example, modified internucleoside linkages such asphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Accordingly, oligonucleotides of thedisclosure can be stabilized against nucleolytic degradation such as bythe incorporation of a modification, e.g., a nucleotide modification.

In some embodiments, an oligonucleotide may be of up to 50 or up to 100nucleotides in length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or morenucleotides of the oligonucleotide are modified nucleotides. Theoligonucleotide may be of 8 to 30 nucleotides in length in which 2 to10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to30 nucleotides of the oligonucleotide are modified nucleotides. Theoligonucleotide may be of 8 to 15 nucleotides in length in which 2 to 4,2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to13, 2 to 14 nucleotides of the oligonucleotide are modified nucleotides.Optionally, the oligonucleotides may have every nucleotide except 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified. Oligonucleotidemodifications are described further herein.

c. Modified Nucleosides

In some embodiments, the oligonucleotide described herein comprises atleast one nucleoside modified at the 2′ position of the sugar. In someembodiments, an oligonucleotide comprises at least one 2′-modifiednucleoside. In some embodiments, all of the nucleosides in theoligonucleotide are 2′-modified nucleosides.

In some embodiments, the oligonucleotide described herein comprises oneor more non-bicyclic 2′-modified nucleosides, e.g., 2′-deoxy, 2′-fluoro(2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl (2′-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′ dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified nucleoside.

In some embodiments, the oligonucleotide described herein comprises oneor more 2′-4′ bicyclic nucleosides in which the ribose ring comprises abridge moiety connecting two atoms in the ring, e.g., connecting the2′-O atom to the 4′-C atom via a methylene (LNA) bridge, an ethylene(ENA) bridge, or a (S)-constrained ethyl (cEt) bridge. Examples of LNAsare described in International Patent Application PublicationWO/2008/043753, published on Apr. 17, 2008, and entitled “RNA AntagonistCompounds For The Modulation Of PCSK9”, the contents of which areincorporated herein by reference in its entirety. Examples of ENAs areprovided in International Patent Publication No. WO 2005/042777,published on May 12, 2005, and entitled “APP/ENA Antisense”; Morita etal., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. GeneTher., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149,2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005;the disclosures of which are incorporated herein by reference in theirentireties. Examples of cEt are provided in U.S. Pat. Nos. 7,101,993;7,399,845 and 7,569,686, each of which is herein incorporated byreference in its entirety.

In some embodiments, the oligonucleotide comprises a modified nucleosidedisclosed in one of the following United States Patent or PatentApplication Publications: U.S. Pat. No. 7,399,845, issued on Jul. 15,2008, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Pat.No. 7,741,457, issued on Jun. 22, 2010, and entitled “6-ModifiedBicyclic Nucleic Acid Analogs”; U.S. Pat. No. 8,022,193, issued on Sep.20, 2011, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S.Pat. No. 7,569,686, issued on Aug. 4, 2009, and entitled “Compounds AndMethods For Synthesis Of Bicyclic Nucleic Acid Analogs”; U.S. Pat. No.7,335,765, issued on Feb. 26, 2008, and entitled “Novel Nucleoside AndOligonucleotide Analogues”; U.S. Pat. No. 7,314,923, issued on Jan. 1,2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”;U.S. Pat. No. 7,816,333, issued on Oct. 19, 2010, and entitled“Oligonucleotide Analogues And Methods Utilizing The Same” and USPublication Number 2011/0009471 now U.S. Pat. No. 8,957,201, issued onFeb. 17, 2015, and entitled “Oligonucleotide Analogues And MethodsUtilizing The Same”, the entire contents of each of which areincorporated herein by reference for all purposes.

In some embodiments, the oligonucleotide comprises at least one modifiednucleoside that results in an increase in Tm of the oligonucleotide in arange of 1° C., 2° C., 3° C., 4° C., or 5° C. compared with anoligonucleotide that does not have the at least one modified nucleoside.The oligonucleotide may have a plurality of modified nucleosides thatresult in a total increase in Tm of the oligonucleotide in a range of 2°C., 3° C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20°C., 25° C., 30° C., 35° C., 40° C., 45° C. or more compared with anoligonucleotide that does not have the modified nucleoside.

The oligonucleotide may comprise a mix of nucleosides of differentkinds. For example, an oligonucleotide may comprise a mix of2′-deoxyribonucleosides or ribonucleosides and 2′-fluoro modifiednucleosides. An oligonucleotide may comprise a mix ofdeoxyribonucleosides or ribonucleosides and 2′-O-Me modifiednucleosides. An oligonucleotide may comprise a mix of 2′-fluoro modifiednucleosides and 2′-O-Me modified nucleosides. An oligonucleotide maycomprise a mix of 2′-4′ bicyclic nucleosides and 2′-MOE, 2′-fluoro, or2′-O-Me modified nucleosides. An oligonucleotide may comprise a mix ofnon-bicyclic 2′-modified nucleosides (e.g., 2′-MOE, 2′-fluoro, or2′-O-Me) and 2′-4′ bicyclic nucleosides (e.g., LNA, ENA, cEt).

The oligonucleotide may comprise alternating nucleosides of differentkinds. For example, an oligonucleotide may comprise alternating2′-deoxyribonucleosides or ribonucleosides and 2′-fluoro modifiednucleosides. An oligonucleotide may comprise alternatingdeoxyribonucleosides or ribonucleosides and 2′-O-Me modifiednucleosides. An oligonucleotide may comprise alternating 2′-fluoromodified nucleosides and 2′-O-Me modified nucleosides. Anoligonucleotide may comprise alternating 2′-4′ bicyclic nucleosides and2′-MOE, 2′-fluoro, or 2′-O-Me modified nucleosides. An oligonucleotidemay comprise alternating non-bicyclic 2′-modified nucleosides (e.g.,2′-MOE, 2′-fluoro, or 2′-O-Me) and 2′-4′ bicyclic nucleosides (e.g.,LNA, ENA, cEt).

In some embodiments, an oligonucleotide described herein comprises a5′-vinylphosphonate modification, one or more abasic residues, and/orone or more inverted abasic residues.

d. Internucleoside Linkages/Backbones

In some embodiments, oligonucleotide may contain a phosphorothioate orother modified internucleoside linkage. In some embodiments, theoligonucleotide comprises phosphorothioate internucleoside linkages. Insome embodiments, the oligonucleotide comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the oligonucleotide comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, oligonucleotides comprise modified internucleoside linkagesat the first, second, and/or (e.g., and) third internucleoside linkageat the 5′ or 3′ end of the nucleotide sequence.

Phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

In some embodiments, oligonucleotides may have heteroatom backbones,such as methylene(methylimino) or MMI backbones; amide backbones (see DeMesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones(see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleicacid (PNA) backbones (wherein the phosphodiester backbone of theoligonucleotide is replaced with a polyamide backbone, the nucleotidesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

e. Stereospecific Oligonucleotides

In some embodiments, internucleotidic phosphorus atoms ofoligonucleotides are chiral, and the properties of the oligonucleotidesby adjusted based on the configuration of the chiral phosphorus atoms.In some embodiments, appropriate methods may be used to synthesizeP-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., asdescribed in Oka N, Wada T, Stereocontrolled synthesis ofoligonucleotide analogs containing chiral internucleotidic phosphorusatoms. Chem Soc Rev. 2011 December; 40(12):5829-43.) In someembodiments, phosphorothioate containing oligonucleotides comprisenucleoside units that are joined together by either substantially all Spor substantially all Rp phosphorothioate intersugar linkages areprovided. In some embodiments, such phosphorothioate oligonucleotideshaving substantially chirally pure intersugar linkages are prepared byenzymatic or chemical synthesis, as described, for example, in U.S. Pat.No. 5,587,261, issued on Dec. 12, 1996, the contents of which areincorporated herein by reference in their entirety. In some embodiments,chirally controlled oligonucleotides provide selective cleavage patternsof a target nucleic acid. For example, in some embodiments, a chirallycontrolled oligonucleotide provides single site cleavage within acomplementary sequence of a nucleic acid, as described, for example, inUS Patent Application Publication 20170037399 A1, published on Feb. 2,2017, entitled “CHIRAL DESIGN”, the contents of which are incorporatedherein by reference in their entirety.

f. Morpholinos

In some embodiments, the oligonucleotide may be a morpholino-basedcompound. Morpholino-based oligomeric compounds are described in DwaineA. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510);Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243,209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra etal., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No.5,034,506, issued Jul. 23, 1991. In some embodiments, themorpholino-based oligomeric compound is a phosphorodiamidate morpholinooligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther.,3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; thedisclosures of which are incorporated herein by reference in theirentireties).

g. Peptide Nucleic Acids (PNAs)

In some embodiments, both a sugar and an internucleoside linkage (thebackbone) of the nucleotide units of an oligonucleotide are replacedwith novel groups. In some embodiments, the base units are maintainedfor hybridization with an appropriate nucleic acid target compound. Onesuch oligomeric compound, an oligonucleotide mimetic that has been shownto have excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, forexample, an aminoethylglycine backbone. The nucleobases are retained andare bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative publication that report thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

h. Gapmers

In some embodiments, an oligonucleotide described herein is a gapmer. Agapmer oligonucleotide generally has the formula 5′-X-Y-Z-3′, with X andZ as flanking regions around a gap region Y. In some embodiments,flanking region X of formula 5′-X-Y-Z-3′ is also referred to as Xregion, flanking sequence X, 5′ wing region X, or 5′ wing segment. Insome embodiments, flanking region Z of formula 5′-X-Y-Z-3′ is alsoreferred to as Z region, flanking sequence Z, 3′ wing region Z, or 3′wing segment. In some embodiments, gap region Y of formula 5′-X-Y-Z-3′is also referred to as Y region, Y segment, or gap-segment Y. In someembodiments, each nucleoside in the gap region Y is a2′-deoxyribonucleoside, and neither the 5′ wing region X or the 3′ wingregion Z contains any 2′-deoxyribonucleosides.

In some embodiments, the Y region is a contiguous stretch ofnucleotides, e.g., a region of 6 or more DNA nucleotides, which arecapable of recruiting an RNAse, such as RNAse H. In some embodiments,the gapmer binds to the target nucleic acid, at which point an RNAse isrecruited and can then cleave the target nucleic acid. In someembodiments, the Y region is flanked both 5′ and 3′ by regions X and Zcomprising high-affinity modified nucleosides, e.g., one to sixhigh-affinity modified nucleosides. Examples of high affinity modifiednucleosides include, but are not limited to, 2′-modified nucleosides(e.g., 2′-MOE, 2′O-Me, 2′-F) or 2′-4′ bicyclic nucleosides (e.g., LNA,cEt, ENA). In some embodiments, the flanking sequences X and Z may be of1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length. Theflanking sequences X and Z may be of similar length or of dissimilarlengths. In some embodiments, the gap-segment Y may be a nucleotidesequence of 5-20 nucleotides, 5-15 twelve nucleotides, or 6-10nucleotides in length.

In some embodiments, the gap region of the gapmer oligonucleotides maycontain modified nucleotides known to be acceptable for efficient RNaseH action in addition to DNA nucleotides, such as C4′-substitutednucleotides, acyclic nucleotides, and arabino-configured nucleotides. Insome embodiments, the gap region comprises one or more unmodifiedinternucleoside linkages. In some embodiments, one or both flankingregions each independently comprise one or more phosphorothioateinternucleoside linkages (e.g., phosphorothioate internucleosidelinkages or other linkages) between at least two, at least three, atleast four, at least five or more nucleotides. In some embodiments, thegap region and two flanking regions each independently comprise modifiedinternucleoside linkages (e.g., phosphorothioate internucleosidelinkages or other linkages) between at least two, at least three, atleast four, at least five or more nucleotides.

A gapmer may be produced using appropriate methods. Representative U.S.patents, U.S. patent publications, and PCT publications that teach thepreparation of gapmers include, but are not limited to, U.S. Pat. Nos.5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922;5,898,031; 7,015,315; 7,101,993; 7,399,845; 7,432,250; 7,569,686;7,683,036; 7,750,131; 8,580,756; 9,045,754; 9,428,534; 9,695,418;10,017,764; 10,260,069; 9,428,534; 8,580,756; U.S. patent publicationNos. US20050074801, US20090221685; US20090286969, US20100197762, andUS20110112170; PCT publication Nos. WO2004069991; WO2005023825;WO2008049085 and WO2009090182; and EP Patent No. EP2,149,605, each ofwhich is herein incorporated by reference in its entirety.

In some embodiments, a gapmer is 10-40 nucleosides in length. Forexample, a gapmer may be 10-40, 10-35, 10-30, 10-25, 10-20, 10-15,15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40,25-35, 25-30, 30-40, 30-35, or 35-40 nucleosides in length. In someembodiments, a gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,or 40 nucleosides in length.

In some embodiments, the gap region Y in a gapmer is 5-20 nucleosides inlength. For example, the gap region Y may be 5-20, 5-15, 5-10, 10-20,10-15, or 15-20 nucleosides in length. In some embodiments, the gapregion Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nucleosides in length. In some embodiments, each nucleoside in the gapregion Y is a 2′-deoxyribonucleoside. In some embodiments, allnucleosides in the gap region Y are 2′-deoxyribonucleosides. In someembodiments, one or more of the nucleosides in the gap region Y is amodified nucleoside (e.g., a 2′ modified nucleoside such as thosedescribed herein). In some embodiments, one or more cytosines in the gapregion Y are optionally 5-methyl-cytosines. In some embodiments, eachcytosine in the gap region Y is a 5-methyl-cytosines.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X-Y-Z-3′formula) and the 3′wing region of a gapmer (Z in the 5′-X-Y-Z-3′formula) are independently 1-20 nucleosides long. For example, the5′wing region of a gapmer (X in the 5′-X-Y-Z-3′ formula) and the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) may be independently1-20, 1-15, 1-10, 1-7, 1-5, 1-3, 1-2, 2-5, 2-7, 3-5, 3-7, 5-20, 5-15,5-10, 10-20, 10-15, or 15-20 nucleosides long. In some embodiments, the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) areindependently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20 nucleosides long. In some embodiments, the 5′wing regionof the gapmer (X in the 5′-X-Y-Z-3′ formula) and the 3′wing region ofthe gapmer (Z in the 5′-X-Y-Z-3′ formula) are of the same length. Insome embodiments, the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) and the 3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′formula) are of different lengths. In some embodiments, the 5′wingregion of the gapmer (X in the 5′-X-Y-Z-3′ formula) is longer than the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula). In someembodiments, the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) is shorter than the 3′wing region of the gapmer (Z in the5′-X-Y-Z-3′ formula).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ of 5-10-5, 4-12-4,3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3,2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1,2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2,1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3,1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2,1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1, 2-14-3, 3-14-2, 1-13-5, 5-13-1,2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3,1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2,2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5,5-14-1, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4,4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1,2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1,1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4,4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7,7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2,3-11-6, 6-11-3, 4-11-5, 5-11-4, 1-19-1, 1-18-2, 2-18-1, 1-17-3, 3-17-1,2-17-2, 1-16-4, 4-16-1, 2-16-3, 3-16-2, 1-15-5, 2-15-4, 4-15-2, 3-15-3,1-14-6, 6-14-1, 2-14-5, 5-14-2, 3-14-4, 4-14-3, 1-13-7, 7-13-1, 2-13-6,6-13-2, 3-13-5, 5-13-3, 4-13-4, 1-12-8, 8-12-1, 2-12-7, 7-12-2, 3-12-6,6-12-3, 4-12-5, 5-12-4, 2-11-8, 8-11-2, 3-11-7, 7-11-3, 4-11-6, 6-11-4,5-11-5, 1-20-1, 1-19-2, 2-19-1, 1-18-3, 3-18-1, 2-18-2, 1-17-4, 4-17-1,2-17-3, 3-17-2, 1-16-5, 2-16-4, 4-16-2, 3-16-3, 1-15-6, 6-15-1, 2-15-5,5-15-2, 3-15-4, 4-15-3, 1-14-7, 7-14-1, 2-14-6, 6-14-2, 3-14-5, 5-14-3,4-14-4, 1-13-8, 8-13-1, 2-13-7, 7-13-2, 3-13-6, 6-13-3, 4-13-5, 5-13-4,2-12-8, 8-12-2, 3-12-7, 7-12-3, 4-12-6, 6-12-4, 5-12-5, 3-11-8, 8-11-3,4-11-7, 7-11-4, 5-11-6, 6-11-5, 1-21-1, 1-20-2, 2-20-1, 1-20-3, 3-19-1,2-19-2, 1-18-4, 4-18-1, 2-18-3, 3-18-2, 1-17-5, 2-17-4, 4-17-2, 3-17-3,1-16-6, 6-16-1, 2-16-5, 5-16-2, 3-16-4, 4-16-3, 1-15-7, 7-15-1, 2-15-6,6-15-2, 3-15-5, 5-15-3, 4-15-4, 1-14-8, 8-14-1, 2-14-7, 7-14-2, 3-14-6,6-14-3, 4-14-5, 5-14-4, 2-13-8, 8-13-2, 3-13-7, 7-13-3, 4-13-6, 6-13-4,5-13-5, 1-12-10, 10-12-1, 2-12-9, 9-12-2, 3-12-8, 8-12- 3, 4-12-7,7-12-4, 5-12-6, 6-12-5, 4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1,1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2,1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4,4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8,8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2,3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4,5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5,6-11-7, or 7-11-6. The numbers indicate the number of nucleosides in X,Y, and Z regions in the 5′-X-Y-Z-3′ gapmer.

In some embodiments, one or more nucleosides in the 5′wing region of agapmer (X in the 5′-X-Y-Z-3′ formula) or the 3′wing region of a gapmer(Z in the 5′-X-Y-Z-3′ formula) are modified nucleotides (e.g.,high-affinity modified nucleosides). In some embodiments, the modifiednucleoside (e.g., high-affinity modified nucleosides) is a 2′-modifiednucleoside. In some embodiments, the 2′-modified nucleoside is a 2′-4′bicyclic nucleoside or a non-bicyclic 2′-modified nucleoside. In someembodiments, the high-affinity modified nucleoside is a 2′-4′ bicyclicnucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2′-modifiednucleoside (e.g., 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA)).

In some embodiments, one or more nucleosides in the 5′wing region of agapmer (X in the 5′-X-Y-Z-3′ formula) are high-affinity modifiednucleosides. In some embodiments, each nucleoside in the 5′wing regionof the gapmer (X in the 5′-X-Y-Z-3′ formula) is a high-affinity modifiednucleoside. In some embodiments, one or more nucleosides in the 3′wingregion of a gapmer (Z in the 5′-X-Y-Z-3′ formula) are high-affinitymodified nucleosides. In some embodiments, each nucleoside in the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) is a high-affinitymodified nucleoside. In some embodiments, one or more nucleosides in the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) arehigh-affinity modified nucleosides and one or more nucleosides in the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) arehigh-affinity modified nucleosides. In some embodiments, each nucleosidein the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) is ahigh-affinity modified nucleoside and each nucleoside in the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) is high-affinitymodified nucleoside.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X-Y-Z-3′formula) comprises the same high affinity nucleosides as the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula). For example, the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) may compriseone or more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or2′-O-Me). In another example, the 5′wing region of the gapmer (X in the5′-X-Y-Z-3′ formula) and the 3′wing region of the gapmer (Z in the5′-X-Y-Z-3′ formula) may comprise one or more 2′-4′ bicyclic nucleosides(e.g., LNA or cEt). In some embodiments, each nucleoside in the 5′wingregion of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula) is a non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me). In some embodiments,each nucleoside in the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) and the 3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′formula) is a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ configuration,wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein each nucleoside in X and Z is anon-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me) and eachnucleoside in Y is a 2′-deoxyribonucleoside. In some embodiments, thegapmer comprises a 5′-X-Y-Z-3′ configuration, wherein X and Z isindependently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in lengthand Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, whereineach nucleoside in X and Z is a 2′-4′ bicyclic nucleosides (e.g., LNA orcEt) and each nucleoside in Y is a 2′-deoxyribonucleoside. In someembodiments, the 5′wing region of the gapmer (X in the 5′-X-Y-Z-3′formula) comprises different high affinity nucleosides as the 3′wingregion of the gapmer (Z in the 5′-X-Y-Z-3′ formula). For example, the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) may compriseone or more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or2′-O-Me) and the 3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′formula) may comprise one or more 2′-4′ bicyclic nucleosides (e.g., LNAor cEt). In another example, the 3′wing region of the gapmer (Z in the5′-X-Y-Z-3′ formula) may comprise one or more non-bicyclic 2′-modifiednucleosides (e.g., 2′-MOE or 2′-O-Me) and the 5′wing region of thegapmer (X in the 5′-X-Y-Z-3′ formula) may comprise one or more 2′-4′bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ configuration,wherein X and Z is independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein each nucleoside in X is a non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me), each nucleoside in Zis a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), and each nucleosidein Y is a 2′-deoxyribonucleoside. In some embodiments, the gapmercomprises a 5′-X-Y-Z-3′ configuration, wherein X and Z is independently1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10(e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein each nucleosidein X is a 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), each nucleosidein Z is a non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me)and each nucleoside in Y is a 2′-deoxyribonucleoside.

In some embodiments, the 5′wing region of a gapmer (X in the 5′-X-Y-Z-3′formula) comprises one or more non-bicyclic 2′-modified nucleosides(e.g., 2′-MOE or 2′-O-Me) and one or more 2′-4′ bicyclic nucleosides(e.g., LNA or cEt). In some embodiments, the 3′wing region of the gapmer(Z in the 5′-X-Y-Z-3′ formula) comprises one or more non-bicyclic2′-modified nucleosides (e.g., 2′-MOE or 2′-O-Me) and one or more 2′-4′bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the5′wing region of the gapmer (X in the 5′-X-Y-Z-3′ formula) and the3′wing region of the gapmer (Z in the 5′-X-Y-Z-3′ formula) comprise oneor more non-bicyclic 2′-modified nucleosides (e.g., 2′-MOE or 2′ Me) andone or more 2′-4′ bicyclic nucleosides (e.g., LNA or cEt).

In some embodiments, a gapmer comprises a 5′-X-Y-Z-3′ configuration,wherein X and Z is independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7)nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10)nucleosides in length, wherein at least one but not all (e.g., 1, 2, 3,4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X (the 5′ mostposition is position 1) is a non-bicyclic 2′-modified nucleoside (e.g.,2′-MOE or 2′-O-Me), wherein the rest of the nucleosides in both X and Zare 2′-4′ bicyclic nucleosides (e.g., LNA or cEt), and wherein eachnucleoside in Y is a 2′deoxyribonucleoside. In some embodiments, thegapmer comprises a 5′-X-Y-Z-3′ configuration, wherein X and Z isindependently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length andY is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein atleast one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3,4, 5, 6, or 7 in Z (the 5′ most position is position 1) is anon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me), whereinthe rest of the nucleosides in both X and Z are 2′-4′ bicyclicnucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a2′deoxyribonucleoside. In some embodiments, the gapmer comprises a5′-X-Y-Z-3′ configuration, wherein X and Z is independently 2-7 (e.g.,2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8,9, or 10) nucleosides in length, wherein at least one but not all (e.g.,1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X and atleast one of positions but not all (e.g., 1, 2, 3, 4, 5, or 6) 1, 2, 3,4, 5, 6, or 7 in Z (the 5′ most position is position 1) is anon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me), whereinthe rest of the nucleosides in both X and Z are 2′-4′ bicyclicnucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a2′deoxyribonucleoside.

Non-limiting examples of gapmers configurations with a mix ofnon-bicyclic 2′-modified nucleoside (e.g., 2′-MOE or 2′-O-Me) and 2′-4′bicyclic nucleosides (e.g., LNA or cEt) in the 5′wing region of thegapmer (X in the 5′-X-Y-Z-3′ formula) and/or the 3′wing region of thegapmer (Z in the 5′-X-Y-Z-3′ formula) include: BBB-(D)n-BBBAA;KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE;LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA;BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE;LLL-(D)n-LLLEEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA;BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BABA-(D)n-ABAB;KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK;LELE-(D)n-ELEL; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL;BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; ABAB-(D)n-ABAB;AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK;ELEL-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL;EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; AABB-(D)n-BBAA;BBAA-(D)n-AABB; AAKK-(D)n-KKAA; AALL-(D)n-LLAA; EEBB-(D)n-BBEE;EEKK-(D)n-KKEE; EELL-(D)n-LLEE; AABB-(D)n-BBAA; AAKK-(D)n-KKAA;AALL-(D)n-LLAA; EEBB-(D)n-BBEE; EEKK-(D)n-KKEE; EELL-(D)n-LLEE;BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA; BBB-(D)n-BBE; KKK-(D)n-KKE;LLL-(D)n-LLE; BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA; BBB-(D)n-BBE;KKK-(D)n-KKE; LLL-(D)n-LLE; BBB-(D)n-BBA; KKK-(D)n-KKA; LLL-(D)n-LLA;BBB-(D)n-BBE; KKK-(D)n-KKE; LLL-(D)n-LLE; ABBB-(D)n-BBBA;AKKK-(D)n-KKKA; ALLL-(D)n-LLLA; EBBB-(D)n-BBBE; EKKK-(D)n-KKKE;ELLL-(D)n-LLLE; ABBB-(D)n-BBBA; AKKK-(D)n-KKKA; ALLL-(D)n-LLLA;EBBB-(D)n-BBBE; EKKK-(D)n-KKKE; ELLL-(D)n-LLLE; ABBB-(D)n-BBBAA;AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA; EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE;ELLL-(D)n-LLLEE; ABBB-(D)n-BBBAA; AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE; ELLL-(D)n-LLLEE; AABBB-(D)n-BBB;AAKKK-(D)n-KKK; AALLL-(D)n-LLL; EEBBB-(D)n-BBB; EEKKK-(D)n-KKK;EELLL-(D)n-LLL; AABBB-(D)n-BBB; AAKKK-(D)n-KKK; AALLL-(D)n-LLL;EEBBB-(D)n-BBB; EEKKK-(D)n-KKK; EELLL-(D)n-LLL; AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA; AALLL-(D)n-LLLA; EEBBB-(D)n-BBBE; EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE; AABBB-(D)n-BBBA; AAKKK-(D)n-KKKA; AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE; EEKKK-(D)n-KKKE; EELLL-(D)n-LLLE; ABBAABB-(D)n-BB;AKKAAKK-(D)n-KK; ALLAALLL-(D)n-LL; EBBEEBB-(D)n-BB; EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL; ABBAABB-(D)n-BB; AKKAAKK-(D)n-KK; ALLAALL-(D)n-LL;EBBEEBB-(D)n-BB; EKKEEKK-(D)n-KK; ELLEELL-(D)n-LL; ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK; ALLALLL-(D)n-LLL; EBBEBB-(D)n-BBB; EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL; ABBABB-(D)n-BBB; AKKAKK-(D)n-KKK; ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB; EKKEKK-(D)n-KKK; ELLELL-(D)n-LLL; EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEE; EK-(D)n-EEEEEEEEEE; EK-(D)n-EEEKK; K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE; K-(D)n-EEKEK; EK-(D)n-EEEEKEKE; EK-(D)n-EEEKEK;EEK-(D)n-KEEKE; EK-(D)n-EEKEK; EK-(D)n-KEEK; EEK-(D)n-EEEKEK;EK-(D)n-KEEEKEE; EK-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK.“A” nucleosides comprise a 2′-modified nucleoside; “B” represents a2′-4′ bicyclic nucleoside; “K” represents a constrained ethyl nucleoside(cEt); “L” represents an LNA nucleoside; and “E” represents a 2′-MOEmodified ribonucleoside; “D” represents a 2′-deoxyribonucleoside; “n”represents the length of the gap segment (Y in the 5′-X-Y-Z-3′configuration) and is an integer between 1-20.

In some embodiments, any one of the gapmers described herein comprisesone or more modified nucleoside linkages (e.g., a phosphorothioatelinkage) in each of the X, Y, and Z regions. In some embodiments, eachinternucleoside linkage in the any one of the gapmers described hereinis a phosphorothioate linkage. In some embodiments, each of the X, Y,and Z regions independently comprises a mix of phosphorothioate linkagesand phosphodiester linkages. In some embodiments, each internucleosidelinkage in the gap region Y is a phosphorothioate linkage, the 5′wingregion X comprises a mix of phosphorothioate linkages and phosphodiesterlinkages, and the 3′wing region Z comprises a mix of phosphorothioatelinkages and phosphodiester linkages.

i. Mixmers

In some embodiments, an oligonucleotide described herein may be a mixmeror comprise a mixmer sequence pattern. In general, mixmers areoligonucleotides that comprise both naturally and non-naturallyoccurring nucleosides or comprise two different types of non-naturallyoccurring nucleosides typically in an alternating pattern. Mixmersgenerally have higher binding affinity than unmodified oligonucleotidesand may be used to specifically bind a target molecule, e.g., to block abinding site on the target molecule. Generally, mixmers do not recruitan RNase to the target molecule and thus do not promote cleavage of thetarget molecule. Such oligonucleotides that are incapable of recruitingRNase H have been described, for example, see WO2007/112754 orWO2007/112753.

In some embodiments, the mixmer comprises or consists of a repeatingpattern of nucleoside analogues and naturally occurring nucleosides, orone type of nucleoside analogue and a second type of nucleosideanalogue. However, a mixmer need not comprise a repeating pattern andmay instead comprise any arrangement of modified nucleoside s andnaturally occurring nucleoside s or any arrangement of one type ofmodified nucleoside and a second type of modified nucleoside. Therepeating pattern, may, for instance be every second or every thirdnucleoside is a modified nucleoside, such as LNA, and the remainingnucleoside s are naturally occurring nucleosides, such as DNA, or are a2′ substituted nucleoside analogue such as 2′-MOE or 2′ fluoroanalogues, or any other modified nucleoside described herein. It isrecognized that the repeating pattern of modified nucleoside, such asLNA units, may be combined with modified nucleoside at fixedpositions—e.g. at the 5′ or 3′ termini.

In some embodiments, a mixmer does not comprise a region of more than 5,more than 4, more than 3, or more than 2 consecutive naturally occurringnucleosides, such as DNA nucleosides. In some embodiments, the mixmercomprises at least a region consisting of at least two consecutivemodified nucleoside, such as at least two consecutive LNAs. In someembodiments, the mixmer comprises at least a region consisting of atleast three consecutive modified nucleoside units, such as at leastthree consecutive LNAs.

In some embodiments, the mixmer does not comprise a region of more than7, more than 6, more than 5, more than 4, more than 3, or more than 2consecutive nucleoside analogues, such as LNAs. In some embodiments, LNAunits may be replaced with other nucleoside analogues, such as thosereferred to herein.

Mixmers may be designed to comprise a mixture of affinity enhancingmodified nucleosides, such as in non-limiting example LNA nucleosidesand 2′-O-Me nucleosides. In some embodiments, a mixmer comprisesmodified internucleoside linkages (e.g., phosphorothioateinternucleoside linkages or other linkages) between at least two, atleast three, at least four, at least five or more nucleosides.

A mixmer may be produced using any suitable method. Representative U.S.patents, U.S. patent publications, and PCT publications that teach thepreparation of mixmers include U.S. patent publication Nos.US20060128646, US20090209748, US20090298916, US20110077288, andUS20120322851, and U.S. Pat. No. 7,687,617.

In some embodiments, a mixmer comprises one or more morpholinonucleosides. For example, in some embodiments, a mixmer may comprisemorpholino nucleosides mixed (e.g., in an alternating manner) with oneor more other nucleosides (e.g., DNA, RNA nucleosides) or modifiednucleosides (e.g., LNA, 2′-O-Me nucleosides).

In some embodiments, mixmers are useful for splice correcting or exonskipping, for example, as reported in Touznik A., et al., LNA/DNAmixmer-based antisense oligonucleotides correct alternative splicing ofthe SMN2 gene and restore SMN protein expression in type 1 SMAfibroblasts Scientific Reports, volume 7, Article number: 3672 (2017),Chen S. et al., Synthesis of a Morpholino Nucleic Acid (MNA)-UridinePhosphoramidite, and Exon Skipping Using MNA/2i-O-Methyl MixmerAntisense Oligonucleotide, Molecules 2016, 21, 1582, the contents ofeach which are incorporated herein by reference.

j. RNA Interference (RNAi)

In some embodiments, oligonucleotides provided herein may be in the formof small interfering RNAs (siRNA), also known as short interfering RNAor silencing RNA. SiRNA, is a class of double-stranded RNA molecules,typically about 20-25 base pairs in length that target nucleic acids(e.g., mRNAs) for degradation via the RNA interference (RNAi) pathway incells. Specificity of siRNA molecules may be determined by the bindingof the antisense strand of the molecule to its target RNA. EffectivesiRNA molecules are generally less than 30 to 35 base pairs in length toprevent the triggering of non-specific RNA interference pathways in thecell via the interferon response, although longer siRNA can also beeffective. In some embodiments, the siRNA molecules are 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, or more base pairs in length. In some embodiments,the siRNA molecules are 8 to 30 base pairs in length, 10 to 15 basepairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs inlength, 19 to 21 base pairs in length, 21 to 23 base pairs in length.

Following selection of an appropriate target RNA sequence, siRNAmolecules that comprise a nucleotide sequence complementary to all or aportion of the target sequence, i.e. an antisense sequence, can bedesigned and prepared using appropriate methods (see, e.g., PCTPublication Number WO 2004/016735; and U.S. Patent Publication Nos.2004/0077574 and 2008/0081791). The siRNA molecule can be doublestranded (i.e. a dsRNA molecule comprising an antisense strand and acomplementary sense strand that hybridizes to form the dsRNA) orsingle-stranded (i.e. a ssRNA molecule comprising just an antisensestrand). The siRNA molecules can comprise a duplex, asymmetric duplex,hairpin or asymmetric hairpin secondary structure, havingself-complementary sense and antisense strands.

In some embodiments, the antisense strand of the siRNA molecule is 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In someembodiments, the antisense strand is 8 to 50 nucleotides in length, 8 to40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 19 to 21 nucleotides in length, 21 to 23nucleotides in lengths.

In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In someembodiments, the sense strand is 8 to 50 nucleotides in length, 8 to 40nucleotides in length, 8 to 30 nucleotides in length, 10 to 15nucleotides in length, 10 to 20 nucleotides in length, 15 to 25nucleotides in length, 19 to 21 nucleotides in length, 21 to 23nucleotides in lengths.

In some embodiments, siRNA molecules comprise an antisense strandcomprising a region of complementarity to a target region in a targetmRNA. In some embodiments, the region of complementarity is at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% complementary to a target region in a targetmRNA. In some embodiments, the target region is a region of consecutivenucleotides in the target mRNA. In some embodiments, a complementarynucleotide sequence need not be 100% complementary to that of its targetto be specifically hybridizable or specific for a target RNA sequence.

In some embodiments, siRNA molecules comprise an antisense strand thatcomprises a region of complementarity to a target RNA sequence and theregion of complementarity is in the range of 8 to 15, 8 to 30, 8 to 40,or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In someembodiments, a region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 nucleotides in length. In some embodiments, the region ofcomplementarity is complementary with at least 6, at least 7, at least8, at least 9, at least 10, at least 11, at least 12, at least 13, atleast 14, at least 15, at least 16, at least 17, at least 18, at least19, at least 20, at least 21, at least 22, at least 23, at least 24, atleast 25 or more consecutive nucleotides of a target RNA sequence. Insome embodiments, siRNA molecules comprise a nucleotide sequence thatcontains no more than 1, 2, 3, 4, or 5 base mismatches compared to theportion of the consecutive nucleotides of target RNA sequence. In someembodiments, siRNA molecules comprise a nucleotide sequence that has upto 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

In some embodiments, siRNA molecules comprise an antisense strandcomprising a nucleotide sequence that is complementary (e.g., at least85%, at least 90%, at least 95%, or 100%) to the target RNA sequence ofthe oligonucleotides provided herein. In some embodiments, siRNAmolecules comprise an antisense strand comprising a nucleotide sequencethat is at least 85%, at least 90%, at least 95%, or 100% identical tothe oligonucleotides provided herein. In some embodiments, siRNAmolecules comprise an antisense strand comprising at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18, at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25 or more consecutive nucleotides of theoligonucleotides provided herein.

Double-stranded siRNA may comprise sense and anti-sense RNA strands thatare the same length or different lengths. Double-stranded siRNAmolecules can also be assembled from a single oligonucleotide in astem-loop structure, wherein self-complementary sense and antisenseregions of the siRNA molecule are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s), as well as circularsingle-stranded RNA having two or more loop structures and a stemcomprising self-complementary sense and antisense strands, wherein thecircular RNA can be processed either in vivo or in vitro to generate anactive siRNA molecule capable of mediating RNAi. Small hairpin RNA(shRNA) molecules thus are also contemplated herein. These moleculescomprise a specific antisense sequence in addition to the reversecomplement (sense) sequence, typically separated by a spacer or loopsequence. Cleavage of the spacer or loop provides a single-stranded RNAmolecule and its reverse complement, such that they may anneal to form adsRNA molecule (optionally with additional processing steps that mayresult in addition or removal of one, two, three or more nucleotidesfrom the 3′ end and/or (e.g., and) the 5′ end of either or bothstrands). A spacer can be of a sufficient length to permit the antisenseand sense sequences to anneal and form a double-stranded structure (orstem) prior to cleavage of the spacer (and, optionally, subsequentprocessing steps that may result in addition or removal of one, two,three, four, or more nucleotides from the 3′ end and/or (e.g., and) the5′ end of either or both strands). A spacer sequence may be an unrelatednucleotide sequence that is situated between two complementarynucleotide sequence regions which, when annealed into a double-strandednucleic acid, comprise a shRNA.

The overall length of the siRNA molecules can vary from about 14 toabout 100 nucleotides depending on the type of siRNA molecule beingdesigned. Generally between about 14 and about 50 of these nucleotidesare complementary to the RNA target sequence, i.e. constitute thespecific antisense sequence of the siRNA molecule. For example, when thesiRNA is a double- or single-stranded siRNA, the length can vary fromabout 14 to about 50 nucleotides, whereas when the siRNA is a shRNA orcircular molecule, the length can vary from about 40 nucleotides toabout 100 nucleotides.

An siRNA molecule may comprise a 3′ overhang at one end of the molecule.The other end may be blunt-ended or have also an overhang (5′ or 3′).When the siRNA molecule comprises an overhang at both ends of themolecule, the length of the overhangs may be the same or different. Inone embodiment, the siRNA molecule of the present disclosure comprises3′ overhangs of about 1 to about 3 nucleotides on both ends of themolecule. In some embodiments, the siRNA molecule comprises 3′ overhangsof about 1 to about 3 nucleotides on the sense strand. In someembodiments, the siRNA molecule comprises 3′ overhangs of about 1 toabout 3 nucleotides on the antisense strand. In some embodiments, thesiRNA molecule comprises 3′ overhangs of about 1 to about 3 nucleotideson both the sense strand and the antisense strand.

In some embodiments, the siRNA molecule comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the siRNA molecule comprises one or more modifiednucleotides and/or (e.g., and) one or more modified internucleotidelinkages. In some embodiments, the modified nucleotide is a modifiedsugar moiety (e.g. a 2′ modified nucleotide). In some embodiments, thesiRNA molecule comprises one or more 2′ modified nucleotides, e.g., a2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), 2′-O-methoxyethyl(2′-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA). In some embodiments, each nucleotide of the siRNA moleculeis a modified nucleotide (e.g., a 2′-modified nucleotide). In someembodiments, the siRNA molecule comprises one or more phosphorodiamidatemorpholinos. In some embodiments, each nucleotide of the siRNA moleculeis a phosphorodiamidate morpholino.

In some embodiments, the siRNA molecule contains a phosphorothioate orother modified internucleotide linkage. In some embodiments, the siRNAmolecule comprises phosphorothioate internucleoside linkages. In someembodiments, the siRNA molecule comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the siRNA molecule comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, the siRNA molecule comprises modified internucleotidelinkages at the first, second, and/or (e.g., and) third internucleosidelinkage at the 5′ or 3′ end of the siRNA molecule.

In some embodiments, the modified internucleotide linkages arephosphorus-containing linkages. In some embodiments,phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Any of the modified chemistries or formats of siRNA molecules describedherein can be combined with each other. For example, one, two, three,four, five, or more different types of modifications can be includedwithin the same siRNA molecule.

In some embodiments, the antisense strand comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the antisense strand comprises one or more modifiednucleotides and/or (e.g., and) one or more modified internucleotidelinkages. In some embodiments, the modified nucleotide comprises amodified sugar moiety (e.g. a 2′ modified nucleotide). In someembodiments, the antisense strand comprises one or more 2′ modifiednucleotides, e.g., a 2′-deoxy, 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me),2′-O-methoxyethyl (2′-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O—NMA). In some embodiments, each nucleotideof the antisense strand is a modified nucleotide (e.g., a 2′-modifiednucleotide). In some embodiments, the antisense strand comprises one ormore phosphorodiamidate morpholinos. In some embodiments, the antisensestrand is a phosphorodiamidate morpholino oligomer (PMO).

In some embodiments, antisense strand contains a phosphorothioate orother modified internucleotide linkage. In some embodiments, theantisense strand comprises phosphorothioate internucleoside linkages. Insome embodiments, the antisense strand comprises phosphorothioateinternucleoside linkages between at least two nucleotides. In someembodiments, the antisense strand comprises phosphorothioateinternucleoside linkages between all nucleotides. For example, in someembodiments, the antisense strand comprises modified internucleotidelinkages at the first, second, and/or (e.g., and) third internucleosidelinkage at the 5′ or 3′ end of the siRNA molecule. In some embodiments,the modified internucleotide linkages are phosphorus-containinglinkages. In some embodiments, phosphorus-containing linkages that maybe used include, but are not limited to, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatescomprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050.

Any of the modified chemistries or formats of the antisense stranddescribed herein can be combined with each other. For example, one, two,three, four, five, or more different types of modifications can beincluded within the same antisense strand.

In some embodiments, the sense strand comprises one or more modifiednucleotides (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In someembodiments, the sense strand comprises one or more modified nucleotidesand/or (e.g., and) one or more modified internucleotide linkages. Insome embodiments, the modified nucleotide is a modified sugar moiety(e.g. a 2′ modified nucleotide). In some embodiments, the sense strandcomprises one or more 2′ modified nucleotides, e.g., a 2′-deoxy,2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me), 2′-0-methoxyethyl (2′-MOE),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O—NMA). In someembodiments, each nucleotide of the sense strand is a modifiednucleotide (e.g., a 2′-modified nucleotide). In some embodiments, thesense strand comprises one or more phosphorodiamidate morpholinos. Insome embodiments, the antisense strand is a phosphorodiamidatemorpholino oligomer (PMO). In some embodiments, the sense strandcontains a phosphorothioate or other modified internucleotide linkage.In some embodiments, the sense strand comprises phosphorothioateinternucleoside linkages. In some embodiments, the sense strandcomprises phosphorothioate internucleoside linkages between at least twonucleotides. In some embodiments, the sense strand comprisesphosphorothioate internucleoside linkages between all nucleotides. Forexample, in some embodiments, the sense strand comprises modifiedinternucleotide linkages at the first, second, and/or (e.g., and) thirdinternucleoside linkage at the 5′ or 3′ end of the sense strand.

In some embodiments, the modified internucleotide linkages arephosphorus-containing linkages. In some embodiments,phosphorus-containing linkages that may be used include, but are notlimited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563, 253; 5,571,799; 5,587,361; and 5,625,050.

Any of the modified chemistries or formats of the sense strand describedherein can be combined with each other. For example, one, two, three,four, five, or more different types of modifications can be includedwithin the same sense strand.

In some embodiments, the antisense or sense strand of the siRNA moleculecomprises modifications that enhance or reduce RNA-induced silencingcomplex (RISC) loading. In some embodiments, the antisense strand of thesiRNA molecule comprises modifications that enhance RISC loading. Insome embodiments, the sense strand of the siRNA molecule comprisesmodifications that reduce RISC loading and reduce off-target effects. Insome embodiments, the antisense strand of the siRNA molecule comprises a2′-O-methoxyethyl (2′-MOE) modification. The addition of the2′-O-methoxyethyl (2′-MOE) group at the cleavage site improves both thespecificity and silencing activity of siRNAs by facilitating theoriented RNA-induced silencing complex (RISC) loading of the modifiedstrand, as described in Song et al., (2017) Mol Ther Nucleic Acids9:242-250, incorporated herein by reference in its entirety. In someembodiments, the antisense strand of the siRNA molecule comprises a2′-OMe-phosphorodithioate modification, which increases RISC loading asdescribed in Wu et al., (2014) Nat Commun 5:3459, incorporated herein byreference in its entirety.

In some embodiments, the sense strand of the siRNA molecule comprises a5′-morpholino, which reduces RISC loading of the sense strand andimproves antisense strand selection and RNAi activity, as described inKumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142, incorporatedherein by reference in its entirety. In some embodiments, the sensestrand of the siRNA molecule is modified with a synthetic RNA-like highaffinity nucleotide analogue, Locked Nucleic Acid (LNA), which reducesRISC loading of the sense strand and further enhances antisense strandincorporation into RISC, as described in Elman et al., (2005) NucleicAcids Res. 33(1): 439-447, incorporated herein by reference in itsentirety. In some embodiments, the sense strand of the siRNA moleculecomprises a 5′ unlocked nucleic acid (UNA) modification, which reduceRISC loading of the sense strand and improve silencing potency of theantisense strand, as described in Snead et al., (2013) Mol Ther NucleicAcids 2(7):e103, incorporated herein by reference in its entirety. Insome embodiments, the sense strand of the siRNA molecule comprises a5-nitroindole modification, which decreased the RNAi potency of thesense strand and reduces off-target effects as described in Zhang etal., (2012) Chembiochem 13(13):1940-1945, incorporated herein byreference in its entirety. In some embodiments, the sense strandcomprises a 2′-O′methyl (2′-O-Me) modification, which reduces RISCloading and the off-target effects of the sense strand, as described inZheng et al., FASEB (2013) 27(10): 4017-4026, incorporated herein byreference in its entirety. In some embodiments, the sense strand of thesiRNA molecule is fully substituted with morpholino, 2′-MOE or 2′-O-Meresidues, and are not recognized by RISC as described in Kole et al.,(2012) Nature reviews. Drug Discovery 11(2):125-140, incorporated hereinby reference in its entirety. In some embodiments the antisense strandof the siRNA molecule comprises a 2′-MOE modification and the sensestrand comprises an 2′-O-Me modification (see e.g., Song et al., (2017)Mol Ther Nucleic Acids 9:242-250). In some embodiments at least one(e.g., at least 2, at least 3, at least 4, at least 5, at least 10)siRNA molecule is linked (e.g., covalently) to a muscle-targeting agent.In some embodiments, the muscle-targeting agent may comprise, or consistof, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), alipid (e.g., a microvesicle), or a sugar moiety (e.g., apolysaccharide). In some embodiments, the muscle-targeting agent is anantibody. In some embodiments, the muscle-targeting agent is ananti-transferrin receptor antibody (e.g., any one of the anti-TfRantibodies provided herein). In some embodiments, the muscle-targetingagent may be linked to the 5′ end of the sense strand of the siRNAmolecule. In some embodiments, the muscle-targeting agent may be linkedto the 3′ end of the sense strand of the siRNA molecule. In someembodiments, the muscle-targeting agent may be linked internally to thesense strand of the siRNA molecule. In some embodiments, themuscle-targeting agent may be linked to the 5′ end of the antisensestrand of the siRNA molecule. In some embodiments, the muscle-targetingagent may be linked to the 3′ end of the antisense strand of the siRNAmolecule. In some embodiments, the muscle-targeting agent may be linkedinternally to the antisense strand of the siRNA molecule.

k. microRNA (miRNAs)

In some embodiments, an oligonucleotide may be a microRNA (miRNA).MicroRNAs (referred to as “miRNAs”) are small non-coding RNAs, belongingto a class of regulatory molecules that control gene expression bybinding to complementary sites on a target RNA transcript. Typically,miRNAs are generated from large RNA precursors (termed pri-miRNAs) thatare processed in the nucleus into approximately 70 nucleotidepre-miRNAs, which fold into imperfect stem-loop structures. Thesepre-miRNAs typically undergo an additional processing step within thecytoplasm where mature miRNAs of 18-25 nucleotides in length are excisedfrom one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer.

As used herein, miRNAs including pri-miRNA, pre-miRNA, mature miRNA orfragments of variants thereof that retain the biological activity ofmature miRNA. In one embodiment, the size range of the miRNA can be from21 nucleotides to 170 nucleotides. In one embodiment the size range ofthe miRNA is from 70 to 170 nucleotides in length. In anotherembodiment, mature miRNAs of from 21 to 25 nucleotides in length can beused.

l. Aptamers

In some embodiments, oligonucleotides provided herein may be in the formof aptamers. Generally, in the context of molecular payloads, aptamer isany nucleic acid that binds specifically to a target, such as a smallmolecule, protein, nucleic acid in a cell. In some embodiments, theaptamer is a DNA aptamer or an RNA aptamer. In some embodiments, anucleic acid aptamer is a single-stranded DNA or RNA (ssDNA or ssRNA).It is to be understood that a single-stranded nucleic acid aptamer mayform helices and/or (e.g., and) loop structures. The nucleic acid thatforms the nucleic acid aptamer may comprise naturally occurringnucleotides, modified nucleotides, naturally occurring nucleotides withhydrocarbon linkers (e.g., an alkylene) or a polyether linker (e.g., aPEG linker) inserted between one or more nucleotides, modifiednucleotides with hydrocarbon or PEG linkers inserted between one or morenucleotides, or a combination of thereof. Exemplary publications andpatents describing aptamers and method of producing aptamers include,e.g., Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Pat. Nos.5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249;5,789,157; 5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCTapplication WO 99/31275, each incorporated herein by reference.

m. Ribozymes

In some embodiments, oligonucleotides provided herein may be in the formof a ribozyme. A ribozyme (ribonucleic acid enzyme) is a molecule,typically an RNA molecule, that is capable of performing specificbiochemical reactions, similar to the action of protein enzymes.Ribozymes are molecules with catalytic activities including the abilityto cleave at specific phosphodiester linkages in RNA molecules to whichthey have hybridized, such as mRNAs, RNA-containing substrates, lncRNAs,and ribozymes, themselves.

Ribozymes may assume one of several physical structures, one of which iscalled a “hammerhead.” A hammerhead ribozyme is composed of a catalyticcore containing nine conserved bases, a double-stranded stem and loopstructure (stem-loop II), and two regions complementary to the targetRNA flanking regions the catalytic core. The flanking regions enable theribozyme to bind to the target RNA specifically by formingdouble-stranded stems I and III. Cleavage occurs in cis (i.e., cleavageof the same RNA molecule that contains the hammerhead motif) or in trans(cleavage of an RNA substrate other than that containing the ribozyme)next to a specific ribonucleotide triplet by a transesterificationreaction from a 3′, 5′-phosphate diester to a 2′, 3′-cyclic phosphatediester. Without wishing to be bound by theory, it is believed that thiscatalytic activity requires the presence of specific, highly conservedsequences in the catalytic region of the ribozyme.

Modifications in ribozyme structure have also included the substitutionor replacement of various non-core portions of the molecule withnon-nucleotidic molecules. For example, Benseler et al. (J. Am. Chem.Soc. (1993) 115:8483-8484) disclosed hammerhead-like molecules in whichtwo of the base pairs of stem II, and all four of the nucleotides ofloop II were replaced with non-nucleoside linkers based on hexaethyleneglycol, propanediol, bis(triethylene glycol) phosphate,tris(propanediol)bisphosphate, or bis(propanediol) phosphate. Ma et al.(Biochem. (1993) 32:1751-1758; Nucleic Acids Res. (1993) 21:2585-2589)replaced the six nucleotide loop of the TAR ribozyme hairpin withnon-nucleotidic, ethylene glycol-related linkers. Thomson et al.(Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear,non-nucleotidic linkers of 13, 17, and 19 atoms in length.

Ribozyme oligonucleotides can be prepared using well known methods (see,e.g., PCT Publications WO9118624; WO9413688; WO9201806; and WO 92/07065;and U.S. Pat. Nos. 5,436,143 and 5,650,502) or can be purchased fromcommercial sources (e.g., US Biochemicals) and, if desired, canincorporate nucleotide analogs to increase the resistance of theoligonucleotide to degradation by nucleases in a cell. The ribozyme maybe synthesized in any known manner, e.g., by use of a commerciallyavailable synthesizer produced, e.g., by Applied Biosystems, Inc. orMilligen. The ribozyme may also be produced in recombinant vectors byconventional means. See, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory (Current edition). The ribozyme RNA sequencesmay be synthesized conventionally, for example, by using RNA polymerasessuch as T7 or SP6.

n. Guide Nucleic Acids

In some embodiments, oligonucleotides are guide nucleic acid, e.g.,guide RNA (gRNA) molecules. Generally, a guide RNA is a short syntheticRNA composed of (1) a scaffold sequence that binds to a nucleic acidprogrammable DNA binding protein (napDNAbp), such as Cas9, and (2) anucleotide spacer portion that defines the DNA target sequence (e.g.,genomic DNA target) to which the gRNA binds in order to bring thenucleic acid programmable DNA binding protein in proximity to the DNAtarget sequence. In some embodiments, the napDNAbp is a nucleicacid-programmable protein that forms a complex with (e.g., binds orassociates with) one or more RNA(s) that targets the nucleicacid-programmable protein to a target DNA sequence (e.g., a targetgenomic DNA sequence). In some embodiments, a nucleic acid-programmablenuclease, when in a complex with an RNA, may be referred to as anuclease:RNA complex. Guide RNAs can exist as a complex of two or moreRNAs, or as a single RNA molecule.

Guide RNAs (gRNAs) that exist as a single RNA molecule may be referredto as single-guide RNAs (sgRNAs), though gRNA is also used to refer toguide RNAs that exist as either single molecules or as a complex of twoor more molecules. Typically, gRNAs that exist as a single RNA speciescomprise two domains: (1) a domain that shares homology to a targetnucleic acid (i.e., directs binding of a Cas9 complex to the target);and (2) a domain that binds a Cas9 protein. In some embodiments, domain(2) corresponds to a sequence known as a tracrRNA and comprises astem-loop structure. In some embodiments, domain (2) is identical orhomologous to a tracrRNA as provided in Jinek et al., Science337:816-821 (2012), the entire contents of which is incorporated hereinby reference.

In some embodiments, a gRNA comprises two or more of domains (1) and(2), and may be referred to as an extended gRNA. For example, anextended gRNA will bind two or more Cas9 proteins and bind a targetnucleic acid at two or more distinct regions, as described herein. ThegRNA comprises a nucleotide sequence that complements a target site,which mediates binding of the nuclease/RNA complex to said target site,providing the sequence specificity of the nuclease:RNA complex. In someembodiments, the RNA-programmable nuclease is the (CRISPR-associatedsystem) Cas9 endonuclease, for example, Cas9 (Csn1) from Streptococcuspyogenes (see, e.g., “Complete genome sequence of an M1 strain ofStreptococcus pyogenes.” Ferretti J. J., McShan W. M., Ajdic D. J.,Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N.,Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G., Najar F. Z., RenQ., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A.,McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNaseIII.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y.,Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature471:602-607 (2011); and “A programmable dual-RNA-guided DNA endonucleasein adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I.,Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), theentire contents of each of which are incorporated herein by reference.

o. Multimers

In some embodiments, molecular payloads may comprise multimers (e.g.,concatemers) of 2 or more oligonucleotides connected by a linker. Inthis way, in some embodiments, the oligonucleotide loading of acomplex/conjugate can be increased beyond the available linking sites ona targeting agent (e.g., available thiol sites or amine sites on anantibody) or otherwise tuned to achieve a particular payload loadingcontent. Oligonucleotides in a multimer can be the same or different(e.g., targeting different genes or different sites on the same gene orproducts thereof).

In some embodiments, multimers comprise 2 or more oligonucleotideslinked together by a cleavable linker. However, in some embodiments,multimers comprise 2 or more oligonucleotides linked together by anon-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4,5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In someembodiments, a multimer comprises 2 to 5, 2 to 10 or 4 to 20oligonucleotides linked together.

In some embodiments, a multimer comprises 2 or more oligonucleotideslinked end-to-end (in a linear arrangement). In some embodiments, amultimer comprises 2 or more oligonucleotides linked end-to-end via anoligonucleotide based linker (e.g., poly-dT linker, an abasic linker).In some embodiments, a multimer comprises a 5′ end of oneoligonucleotide linked to a 3′ end of another oligonucleotide. In someembodiments, a multimer comprises a 3′ end of one oligonucleotide linkedto a 3′ end of another oligonucleotide. In some embodiments, a multimercomprises a 5′ end of one oligonucleotide linked to a 5′ end of anotheroligonucleotide. Still, in some embodiments, multimers can comprise abranched structure comprising multiple oligonucleotides linked togetherby a branching linker.

Further examples of multimers that may be used in the complexes providedherein are disclosed, for example, in US Patent Application Number2015/0315588 A1, entitled Methods of delivering multiple targetingoligonucleotides to a cell using cleavable linkers, which was publishedon Nov. 5, 2015; US Patent Application Number 2015/0247141 A1, entitledMultimeric Oligonucleotide Compounds, which was published on Sep. 3,2015, US Patent Application Number US 2011/0158937 A1, entitledImmunostimulatory Oligonucleotide Multimers, which was published on Jun.30, 2011; and U.S. Pat. No. 5,693,773, entitled Triplex-FormingAntisense Oligonucleotides Having Abasic Linkers Targeting Nucleic AcidsComprising Mixed Sequences Of Purines And Pyrimidines, which issued onDec. 2, 1997, the contents of each of which are incorporated herein byreference in their entireties.

o. Splice Altering Oligonucleotides

In some embodiments, an oligonucleotide (e.g., an antisenseoligonucleotide including a morpholino) of the present disclosure targetsplicing. In some embodiments, the oligonucleotide targets splicing byinducing exon skipping and restoring the reading frame within a gene. Asa non-limiting example, the oligonucleotide may induce skipping of anexon encoding a frameshift mutation and/or (e.g., and) an exon thatencodes a premature stop codon. In some embodiments, an oligonucleotidemay induce exon skipping by blocking spliceosome recognition of a splicesite. In some embodiments, exon skipping results in a truncated butfunctional protein compared to the reference protein (e.g., truncatedbut functional DMD protein as described below). In some embodiments, theoligonucleotide promotes inclusion of a particular exon (e.g., exon 7 ofthe SMN2 gene described below). In some embodiments, an oligonucleotidemay induce inclusion of an exon by targeting a splice site inhibitorysequence. RNA splicing has been implicated in muscle diseases, includingDuchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).

Alterations (e.g., deletions, point mutations, and duplications) in thegene encoding dystrophin (DMD) cause DMD. These alterations can lead toframeshift mutations and/or (e.g., and) nonsense mutations. In someembodiments, an oligonucleotide of the present disclosure promotesskipping of one or more DMD exons (e.g., exon 8, exon 43, exon 44, exon45, exon 50, exon 51, exon 52, exon 53, and/or (e.g., and) exon 55) andresults in a functional truncated protein. See, e.g., U.S. Pat. No.8,486,907 published on Jul. 16, 2013 and U.S. 20140275212 published onSep. 18, 2014.

In SMA, there is loss of functional SMN1. Although the SMN2 gene is aparalog to SMN1, alternative splicing of the SMN2 gene predominantlyleads to skipping of exon 7 and subsequent production of a truncated SMNprotein that cannot compensate for SMN1 loss. In some embodiments, anoligonucleotide of the present disclosure promotes inclusion of SMN2exon 7. In some embodiments, an oligonucleotide is an antisenseoligonucleotide that targets SMN2 splice site inhibitory sequences (see,e.g., U.S. Pat. No. 7,838,657, which was published on Nov. 23, 2010).

ii. Small Molecules:

Any suitable small molecule may be used as a molecular payload, asdescribed herein.

iii. Peptides/Proteins

Any suitable peptide or protein may be used as a molecular payload, asdescribed herein. In some embodiments, a protein is an enzyme (e.g., anacid alpha-glucosidase, e.g., as encoded by the GAA gene). Thesepeptides or proteins may be produced, synthesized, and/or (e.g., and)derivatized using several methodologies, e.g. phage displayed peptidelibraries, one-bead one-compound peptide libraries, or positionalscanning synthetic peptide combinatorial libraries. Exemplarymethodologies have been characterized in the art and are incorporated byreference (Gray, B. P. and Brown, K. C. “Combinatorial PeptideLibraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114:2,1020-1081.; Samoylova, T. I. and Smith, B. F. “Elucidation ofmuscle-binding peptides by phage display screening.” Muscle Nerve, 1999,22:4. 460-6.).

iv. Nucleic Acid Constructs

Any suitable gene expression construct may be used as a molecularpayload, as described herein. In some embodiments, a gene expressionconstruct may be a vector or a cDNA fragment. In some embodiments, agene expression construct may be messenger RNA (mRNA). In someembodiments, a mRNA used herein may be a modified mRNA, e.g., asdescribed in U.S. Pat. No. 8,710,200, issued on Apr. 24, 2014, entitled“Engineered nucleic acids encoding a modified erythropoietin and theirexpression”. In some embodiments, a mRNA may comprise a 5′-methyl cap.In some embodiments, a mRNA may comprise a polyA tail, optionally of upto 160 nucleotides in length. A gene expression construct may encode asequence of a protein that is deficient in a muscle disease. In someembodiments, the gene expression construct may be expressed, e.g.,overexpressed, within the nucleus of a muscle cell. In some embodiments,the gene expression construct encodes a gene that is deficient in amuscle disease. In some embodiments, the gene expression constructencodes a protein that comprises at least one zinc finger. In someembodiments, the gene expression construct encodes a protein that bindsto a gene in Table 6. In some embodiments, the gene expression constructencodes a protein that leads to a reduction in the expression of aprotein (e.g., mutant protein) encoded by a gene in Table 6. In someembodiments, the gene expression construct encodes a gene editingenzyme. Additional examples of nucleic acid constructs that may be usedas molecular payloads are provided in International Patent ApplicationPublication WO2017152149A1, published on Sep. 19, 2017, entitled,“CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER”; U.S. Pat.No. 8,853,377B2, issued on Oct. 7, 2014, entitled, “MRNA FOR USE INTREATMENT OF HUMAN GENETIC DISEASES”; and US Patent U.S. Pat. No.8,822,663B2, issued on Sep. 2, 2014, ENGINEERED NUCLEIC ACIDS ANDMETHODS OF USE THEREOF,” the contents of each of which are incorporatedherein by reference in their entireties.

v. Detectable Labels/Diagnostic Agents

Any suitable detectable label or diagnostic agent can be used as themolecular payload of the present disclosure. A “diagnostic agent” refersto an agent that is used for diagnostic purpose, e.g., by detectinganother molecule in a cell or a tissue. In some embodiments, thediagnostic agent is an agent that targets (e.g., binds) a biomarkerknown to be associated with a disease (e.g., a nucleic acid biomarker,protein biomarker, or a metabolite biomarker) in a subject and producesa detectable signal, which can be used to determine the presence/absenceof the biomarker, thus to diagnose a disease. For example, thediagnostic agent may be, without limitation, an antibody or an antisensenucleic acid.

In some embodiments, the diagnostic agent contains a detectable label. Adetectable label refers to a moiety that has at least one element,isotope, or a structural or functional group incorporated that enablesdetection of a molecule, e.g., a protein or polypeptide, or otherentity, to which the diagnostic agent binds. In some embodiments, adetectable label falls into any one (or more) of five classes: a) anagent which contains isotopic moieties, which may be radioactive orheavy isotopes, including, but not limited to, 2H, 3H, 13C, 14C, 15N,18F, 31P, 32P, 35S, 67Ga, 76Br, 99mTc (Tc-99m), 111In, 123I, 125I, 131I,153Gd, 169Yb, and 186Re; b) an agent which contains an immune moiety,which may be an antibody or antigen, which may be bound to an enzyme(e.g., such as horseradish peroxidase); c) an agent comprising acolored, luminescent, phosphorescent, or fluorescent moiety (e.g., suchas the fluorescent label fluorescein isothiocyanate (FITC); d) an agentwhich has one or more photo affinity moieties; and e) an agent which isa ligand for one or more known binding partners (e.g.,biotin-streptavidin, His-NiTNAFK506-FKBP). In some embodiments, adetectable label comprises a radioactive isotope. In some embodiments, adetectable label comprises a fluorescent moiety. In some embodiments,the detectable label comprises a dye, e.g., a fluorescent dye, e.g.,fluorescein isothiocyanate, Texas red, rhodamine, Cy3, Cy5, Cy5.5, Alexa647 and derivatives. In some embodiments, the detectable label comprisesbiotin. In some embodiments, the detectable molecule is a fluorescentpolypeptide (e.g., GFP or a derivative thereof such as enhanced GFP(EGFP)) or a luciferase (e.g., a firefly, Renilla, or Gaussialuciferase). In some embodiments, a detectable label may react with asuitable substrate (e.g., a luciferin) to generate a detectable signal.Non-limiting examples of fluorescent proteins include GFP andderivatives thereof, proteins comprising chromophores that emit light ofdifferent colors such as red, yellow, and cyan fluorescent proteins,etc. Exemplary fluorescent proteins include, e.g., Sirius, Azurite,EBFP2, TagBFP, mTurquoise, ECFP, Cerulean, TagCFP, mTFP1, mUkG1, mAG1,AcGFP1, TagGFP2, EGFP, mWasabi, EmGFP, TagYPF, EYFP, Topaz, SYFP2,Venus, Citrine, mKO, mKO2, mOrange, mOrange2, TagRFP, TagRFP-T,mStrawberry, mRuby, mCherry, mRaspberry, mKate2, mPlum, mNeptune,T-Sapphire, mAmetrine, mKeima. See, e.g., Chalfie, M. and Kain, S R(eds.) Green fluorescent protein: properties, applications, andprotocols (Methods of biochemical analysis, v. 47, Wiley-Interscience,and Hoboken, N.J., 2006, and/or (e.g., and) Chudakov, D M, et al.,Physiol Rev. 90(3):1103-63, 2010, incorporated herein by reference, fordiscussion of GFP and numerous other fluorescent or luminescentproteins. In some embodiments, a detectable label comprises a darkquencher, e.g., a substance that absorbs excitation energy from afluorophore and dissipates the energy as heat.

B. Linkers

Complexes described herein generally comprise a linker that connects anyone of the anti-TfR antibodies described herein to a molecular payload.A linker comprises at least one covalent bond. In some embodiments, alinker may be a single bond, e.g., a disulfide bond or disulfide bridge,that connects an anti-TfR antibody to a molecular payload. However, insome embodiments, a linker may connect any one of the anti-TfRantibodies described herein to a molecular payload through multiplecovalent bonds. In some embodiments, a linker may be a cleavable linker.However, in some embodiments, a linker may be a non-cleavable linker. Alinker is generally stable in vitro and in vivo, and may be stable incertain cellular environments. Additionally, generally a linker does notnegatively impact the functional properties of either the anti-TfRantibody or the molecular payload. Examples and methods of synthesis oflinkers are known in the art (see, e.g. Kline, T. et al. “Methods toMake Homogenous Antibody Drug Conjugates.” Pharmaceutical Research,2015, 32:11, 3480-3493.; Jain, N. et al. “Current ADC Linker Chemistry”Pharm Res. 2015, 32:11, 3526-3540.; McCombs, J. R. and Owen, S. C.“Antibody Drug Conjugates: Design and Selection of Linker, Payload andConjugation Chemistry” AAPS J. 2015, 17:2, 339-351.).

A precursor to a linker typically will contain two different reactivespecies that allow for attachment to both the anti-TfR antibody and amolecular payload. In some embodiments, the two different reactivespecies may be a nucleophile and/or (e.g., and) an electrophile. In someembodiments, a linker is connected to an anti-TfR antibody viaconjugation to a lysine residue or a cysteine residue of the anti-TfRantibody. In some embodiments, a linker is connected to a cysteineresidue of an anti-TfR antibody via a maleimide-containing linker,wherein optionally the maleimide-containing linker comprises amaleimidocaproyl or maleimidomethyl cyclohexane-1-carboxylate group. Insome embodiments, a linker is connected to a cysteine residue of ananti-TfR antibody or thiol functionalized molecular payload via a3-arylpropionitrile functional group. In some embodiments, a linker isconnected to a lysine residue of an anti-TfR antibody. In someembodiments, a linker is connected to an anti-TfR antibody and/or (e.g.,and) a molecular payload via an amide bond, a carbamate bond, ahydrazide, a trizaole, a thioether, or a disulfide bond.

i. Cleavable Linkers

A cleavable linker may be a protease-sensitive linker, a pH-sensitivelinker, or a glutathione-sensitive linker. These linkers are generallycleavable only intracellularly and are preferably stable inextracellular environments, e.g. extracellular to a muscle cell.

Protease-sensitive linkers are cleavable by protease enzymatic activity.These linkers typically comprise peptide sequences and may be 2-10 aminoacids, about 2-5 amino acids, about 5-10 amino acids, about 10 aminoacids, about 5 amino acids, about 3 amino acids, or about 2 amino acidsin length. In some embodiments, a peptide sequence may comprisenaturally-occurring amino acids, e.g. cysteine, alanine, ornon-naturally-occurring or modified amino acids. Non-naturally occurringamino acids include β-amino acids, homo-amino acids, prolinederivatives, 3-substituted alanine derivatives, linear core amino acids,N-methyl amino acids, and others known in the art. In some embodiments,a protease-sensitive linker comprises a valine-citrulline oralanine-citrulline dipeptide sequence. In some embodiments, aprotease-sensitive linker can be cleaved by a lysosomal protease, e.g.cathepsin B, and/or (e.g., and) an endosomal protease.

A pH-sensitive linker is a covalent linkage that readily degrades inhigh or low pH environments. In some embodiments, a pH-sensitive linkermay be cleaved at a pH in a range of 4 to 6. In some embodiments, apH-sensitive linker comprises a hydrazone or cyclic acetal. In someembodiments, a pH-sensitive linker is cleaved within an endosome or alysosome.

In some embodiments, a glutathione-sensitive linker comprises adisulfide moiety. In some embodiments, a glutathione-sensitive linker iscleaved by a disulfide exchange reaction with a glutathione speciesinside a cell. In some embodiments, the disulfide moiety furthercomprises at least one amino acid, e.g. a cysteine residue.

In some embodiments, the linker is a Val-cit linker (e.g., as describedin U.S. Pat. No. 6,214,345, incorporated herein by reference). In someembodiments, before conjugation, the val-cit linker has a structure of:

In some embodiments, after conjugation, the val-cit linker has astructure of:

In some embodiments, the Val-cit linker is attached to a reactivechemical moiety (e.g., SPAAC for click chemistry conjugation). In someembodiments, before click chemistry conjugation, the val-cit linkerattached to a reactive chemical moiety (e.g., SPAAC for click chemistryconjugation) has the structure of:

wherein n is any number from 0-10. In some embodiments, n is 3.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated(e.g., via a different chemical moiety) to a molecular payload (e.g., anoligonucleotide). In some embodiments, the val-cit linker attached to areactive chemical moiety (e.g., SPAAC for click chemistry conjugation)and conjugated to a molecular payload (e.g., an oligonucleotide) has thestructure of (before click chemistry conjugation):

wherein n is any number from 0-10. In some embodiments, n is 3.

In some embodiments, after conjugation to a molecular payload (e.g., anoligonucleotide), the val-cit linker has a structure of:

wherein n is any number from 0-10, and wherein m is any number from0-10. In some embodiments, n is 3 and m is 4.

ii. Non-Cleavable Linkers

In some embodiments, non-cleavable linkers may be used. Generally, anon-cleavable linker cannot be readily degraded in a cellular orphysiological environment. In some embodiments, a non-cleavable linkercomprises an optionally substituted alkyl group, wherein thesubstitutions may include halogens, hydroxyl groups, oxygen species, andother common substitutions. In some embodiments, a linker may comprisean optionally substituted alkyl, an optionally substituted alkylene, anoptionally substituted arylene, a heteroarylene, a peptide sequencecomprising at least one non-natural amino acid, a truncated glycan, asugar or sugars that cannot be enzymatically degraded, an azide, analkyne-azide, a peptide sequence comprising a LPXT sequence, athioether, a biotin, a biphenyl, repeating units of polyethylene glycolor equivalent compounds, acid esters, acid amides, sulfamides, and/or(e.g., and) an alkoxy-amine linker. In some embodiments,sortase-mediated ligation will be utilized to covalently link ananti-TfR antibody comprising a LPXT sequence to a molecular payloadcomprising a (G)_(n) sequence (see, e.g. Proft T. Sortase-mediatedprotein ligation: an emerging biotechnology tool for proteinmodification and immobilization. Biotechnol Lett. 2010, 32(1):1-10.).

In some embodiments, a linker may comprise a substituted alkylene, anoptionally substituted alkenylene, an optionally substituted alkynylene,an optionally substituted cycloalkylene, an optionally substitutedcycloalkenylene, an optionally substituted arylene, an optionallysubstituted heteroarylene further comprising at least one heteroatomselected from N, O, and S; an optionally substituted heterocyclylenefurther comprising at least one heteroatom selected from N, O, and S; animino, an optionally substituted nitrogen species, an optionallysubstituted oxygen species O, an optionally substituted sulfur species,or a poly(alkylene oxide), e.g. polyethylene oxide or polypropyleneoxide.

iii. Linker Conjugation

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload via a phosphate, thioether, ether,carbon-carbon, carbamate, or amide bond. In some embodiments, a linkeris connected to an oligonucleotide through a phosphate orphosphorothioate group, e.g. a terminal phosphate of an oligonucleotidebackbone. In some embodiments, a linker is connected to an anti-TfRantibody, through a lysine or cysteine residue present on the anti-TfRantibody.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by a cycloaddition reaction betweenan azide and an alkyne to form a triazole, wherein the azide and thealkyne may be located on the anti-TfR antibody, molecular payload, orthe linker. In some embodiments, an alkyne may be a cyclic alkyne, e.g.,a cyclooctyne. In some embodiments, an alkyne may be bicyclononyne (alsoknown as bicyclo[6.1.0]nonyne or BCN) or substituted bicyclononyne. Insome embodiments, a cyclooctane is as described in International PatentApplication Publication WO2011136645, published on Nov. 3, 2011,entitled, “Fused Cyclooctyne Compounds And Their Use In Metal-free ClickReactions”. In some embodiments, an azide may be a sugar or carbohydratemolecule that comprises an azide. In some embodiments, an azide may be6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In someembodiments, a sugar or carbohydrate molecule that comprises an azide isas described in International Patent Application PublicationWO2016170186, published on Oct. 27, 2016, entitled, “Process For TheModification Of A Glycoprotein Using A Glycosyltransferase That Is Or IsDerived From A β(1,4)-N-Acetylgalactosaminyltransferase”. In someembodiments, a cycloaddition reaction between an azide and an alkyne toform a triazole, wherein the azide and the alkyne may be located on theanti-TfR antibody, molecular payload, or the linker is as described inInternational Patent Application Publication WO2014065661, published onMay 1, 2014, entitled, ‘Modified antibody, antibody-conjugate andprocess for the preparation thereof’; or International PatentApplication Publication WO2016170186, published on Oct. 27, 2016,entitled, “Process For The Modification Of A Glycoprotein Using AGlycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”.

In some embodiments, a linker further comprises a spacer, e.g., apolyethylene glycol spacer or an acyl/carbomoyl sulfamide spacer, e.g.,a HydraSpace™ spacer. In some embodiments, a spacer is as described inVerkade, J. M. M. et al., “A Polar Sulfamide Spacer SignificantlyEnhances the Manufacturability, Stability, and Therapeutic Index ofAntibody- Drug Conjugates”, Antibodies, 2018, 7, 12.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by the Diels-Alder reaction betweena dienophile and a diene/hetero-diene, wherein the dienophile and thediene/hetero-diene may be located on the anti-TfR antibody, molecularpayload, or the linker. In some embodiments a linker is connected to ananti-TfR antibody and/or (e.g., and) molecular payload by otherpericyclic reactions, e.g. ene reaction. In some embodiments, a linkeris connected to an anti-TfR antibody and/or (e.g., and) molecularpayload by an amide, thioamide, or sulfonamide bond reaction. In someembodiments, a linker is connected to an anti-TfR antibody and/or (e.g.,and) molecular payload by a condensation reaction to form an oxime,hydrazone, or semicarbazide group existing between the linker and theanti-TfR antibody and/or (e.g., and) molecular payload.

In some embodiments, a linker is connected to an anti-TfR antibodyand/or (e.g., and) molecular payload by a conjugate addition reactionbetween a nucleophile, e.g. an amine or a hydroxyl group, and anelectrophile, e.g. a carboxylic acid or an aldehyde. In someembodiments, a nucleophile may exist on a linker and an electrophile mayexist on an anti-TfR antibody or molecular payload prior to a reactionbetween a linker and an anti-TfR antibody or molecular payload. In someembodiments, an electrophile may exist on a linker and a nucleophile mayexist on an anti-TfR antibody or molecular payload prior to a reactionbetween a linker and an anti-TfR antibody or molecular payload. In someembodiments, an electrophile may be an azide, pentafluorophenyl, asilicon centers, a carbonyl, a carboxylic acid, an anhydride, anisocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidylester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide,an episulfide, an aziridine, an aryl, an activated phosphorus center,and/or (e.g., and) an activated sulfur center. In some embodiments, anucleophile may be an optionally substituted alkene, an optionallysubstituted alkyne, an optionally substituted aryl, an optionallysubstituted heterocyclyl, a hydroxyl group, an amino group, analkylamino group, an anilido group, or a thiol group.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated tothe anti-TfR antibody by a structure of:

wherein m is any number from 0-10. In some embodiments, m is 4.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) is conjugated to ananti-TfR antibody having a structure of:

wherein m is any number from 0-10. In some embodiments, m is 4.

In some embodiments, the val-cit linker attached to a reactive chemicalmoiety (e.g., SPAAC for click chemistry conjugation) and conjugated toan anti-TfR antibody has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4.

In some embodiments, the val-cit linker that links the antibody and themolecular payload has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments,n is 3 and/or (e.g., and) m is 4. In some embodiments, X is NH (e.g., NHfrom an amine group of a lysine), S (e.g., S from a thiol group of acysteine), or O (e.g., 0 from a hydroxyl group of a serine, threonine,or tyrosine) of the antibody.

In some embodiments, the complex described herein has a structure of:

wherein n is any number from 0-10, wherein m is any number from 0-10. Insome embodiments, n is 3 and/or (e.g., and) m is 4.

In structures formula (A), (B), (C), and (D), L1, in some embodiments,is a spacer that is a substituted or unsubstituted aliphatic,substituted or unsubstituted heteroaliphatic, substituted orunsubstituted carbocyclylene, substituted or unsubstitutedheterocyclylene, substituted or unsubstituted arylene, substituted orunsubstituted heteroarylene, —O—, —N(R^(A))—, —S—, —C(═O)—, —C(═O)O—,—C(═O)NR^(A)—, —NR^(A)C(═O)—, —NR^(A)C(═O)R^(A)—, —C(═O)R^(A)—,—NR^(A)C(═O)O—, —NR^(A)C(═O)N(R^(A))—, —OC(═O)—, —OC(═O)O—,—OC(═O)N(R^(A))—, —S(O)₂NR^(A)—, —NR^(A)S(O)₂—, or a combinationthereof. In some embodiments, L1 is

wherein the piperazine moiety links to the oligonucleotide, wherein L2is

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide.

In some embodiments, L1 is

In some embodiments, L1 is linked to a 5′ phosphate of theoligonucleotide.

In some embodiments, L1 is optional (e.g., need not be present).

In some embodiments, any one of the complexes described herein has astructure of:

C. Examples of Antibody-Molecular Payload Complexes

Further provided herein are non-limiting examples of complexescomprising any one the anti-TfR antibodies described herein covalentlylinked to any of the molecular payloads (e.g., an oligonucleotide)described herein. In some embodiments, the anti-TfR antibody (e.g., anyone of the anti-TfR antibody provided in Table 2) is covalently linkedto a molecular payload (e.g., an oligonucleotide) via a linker. Any ofthe linkers described herein may be used. In some embodiments, if themolecular payload is an oligonucleotide, the linker is linked to the 5′end, the 3′ end, or internally of the oligonucleotide. In someembodiments, the linker is linked to the anti-TfR antibody via athiol-reactive linkage (e.g., via a cysteine in the anti-TfR antibody).In some embodiments, the linker (e.g., a Val-cit linker) is linked tothe antibody (e.g., an anti-TfR antibody described herein) via an aminegroup (e.g., via a lysine in the antibody).

An example of a structure of a complex comprising an anti-TfR antibodycovalently linked to a molecular payload via a Val-cit linker isprovided below:

wherein the linker is linked to the antibody via a thiol-reactivelinkage (e.g., via a cysteine in the antibody).

Another example of a structure of a complex comprising an anti-TfRantibody covalently linked to a molecular payload via a Val-cit linkeris provided below:

wherein n is a number between 0-10, wherein m is a number between 0-10,wherein the linker is linked to the antibody via an amine group (e.g.,on a lysine residue), and/or (e.g., and) wherein the linker is linked tothe oligonucleotide (e.g., at the 5′ end, 3′ end, or internally). Insome embodiments, the linker is linked to the antibody via a lysine, thelinker is linked to the oligonucleotide at the 5′ end, n is 3, and m is4. In some embodiments, the molecular payload is an oligonucleotidecomprising a sense strand and an antisense strand, and the linker islinked to the sense strand or the antisense strand at the 5′ end or the3′ end.

It should be appreciated that antibodies can be linked to molecularpayloads with different stoichiometries, a property that may be referredto as a drug to antibody ratios (DAR) with the “drug” being themolecular payload. In some embodiments, one molecular payload is linkedto an antibody (DAR=1). In some embodiments, two molecular payloads arelinked to an antibody (DAR=2). In some embodiments, three molecularpayloads are linked to an antibody (DAR=3). In some embodiments, fourmolecular payloads are linked to an antibody (DAR=4). In someembodiments, a mixture of different complexes, each having a differentDAR, is provided. In some embodiments, an average DAR of complexes insuch a mixture may be in a range of 1 to 3, 1 to 4, 1 to 5 or more. DARmay be increased by conjugating molecular payloads to different sites onan antibody and/or (e.g., and) by conjugating multimers to one or moresites on antibody. For example, a DAR of 2 may be achieved byconjugating a single molecular payload to two different sites on anantibody or by conjugating a dimer molecular payload to a single site ofan antibody.

In some embodiments, the complex described herein comprises an anti-TfRantibody described herein (e.g., antibodies in Tables 2-5) covalentlylinked to a molecular payload. In some embodiments, the complexdescribed herein comprises an anti-TfR antibody described herein (e.g.,antibodies in Tables 2-5) covalently linked to molecular payload via alinker (e.g., a Val-cit linker). In some embodiments, the linker (e.g.,a Val-cit linker) is linked to the antibody (e.g., an anti-TfR antibodydescribed herein) via a thiol-reactive linkage (e.g., via a cysteine inthe antibody). In some embodiments, the linker (e.g., a Val-cit linker)is linked to the antibody (e.g., an anti-TfR antibody described herein)via an amine group (e.g., via a lysine in the antibody).

In some embodiments, in any one of the examples of complexes describedherein, the molecular payload is an oligonucleotide comprising a regionof complementarily of at least 15 nucleotides to any one of the genetarget sequences described herein.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a CDR-H1, a CDR-H2, and a CDR-H3 that are the same asthe CDR-H1, CDR-H2, and CDR-H3 shown in Table 2; and a CDR-L1, a CDR-L2,and a CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shownin Table 2.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acidsequence of SEQ ID NO: 70.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQID NO: 74.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQID NO: 75.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:77, and a VL comprising the amino acid sequence of SEQ ID NO: 78.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a VH comprising the amino acid sequence of SEQ ID NO:77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQID NO: 80.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 84, SEQ ID NO: 86 or SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the aminoacid sequence of SEQ ID NO: 89.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the aminoacid sequence of SEQ ID NO: 90.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 92 or SEQ ID NO: 94, and a light chain comprising the aminoacid sequence of SEQ ID NO: 95.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 92, and a light chain comprising the amino acid sequence ofSEQ ID NO: 93.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99 and a VL comprising theamino acid sequence of SEQ ID NO: 85.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 89.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 100 or SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 90.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 102 and a light chain comprising the amino acid sequence ofSEQ ID NO: 93.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked to a molecular payload, wherein the anti-TfRantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 102 or SEQ ID NO: 103 and a light chain comprising the aminoacid sequence of SEQ ID NO: 95.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 84 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 86 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 87 and a light chaincomprising the amino acid sequence of SEQ ID NO: 85; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 89; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 88 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of n SEQ ID NO: 89; wherein thecomplex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 91 and a light chaincomprising the amino acid sequence of SEQ ID NO: 90; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 93; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 94 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRantibody covalently linked via a lysine to the 5′ end of anoligonucleotide, wherein the anti-TfR antibody comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 92 and a light chaincomprising the amino acid sequence of SEQ ID NO: 95; wherein the complexhas the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 69 and a VL comprising the amino acid sequence ofSEQ ID NO: 70; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 71 and a VL comprising the amino acid sequence ofSEQ ID NO: 70; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 72 and a VL comprising the amino acid sequence ofSEQ ID NO: 70; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence ofSEQ ID NO: 74; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 73 and a VL comprising the amino acid sequence ofSEQ ID NO: 75; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence ofSEQ ID NO: 74; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence ofSEQ ID NO: 75; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence ofSEQ ID NO: 78; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 79 and a VL comprising the amino acid sequence ofSEQ ID NO: 80; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a VH comprising the amino acidsequence of SEQ ID NO: 77 and a VL comprising the amino acid sequence ofSEQ ID NO: 80; wherein the complex has the structure of:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 97 and a light chain comprising the aminoacid sequence of SEQ ID NO: 85; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 98 and a light chain comprising the aminoacid sequence of SEQ ID NO: 85; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 99 and a light chain comprising the aminoacid sequence of SEQ ID NO: 85; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 100 and a light chain comprising the aminoacid sequence of i SEQ ID NO: 89; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 100 and a light chain comprising the aminoacid sequence of SEQ ID NO: 90; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 89; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 101 and a light chain comprising the aminoacid sequence of SEQ ID NO: 90; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 102 and a light chain comprising the aminoacid sequence of SEQ ID NO: 93; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 103 and a light chain comprising the aminoacid sequence of SEQ ID NO: 95; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, the complex described herein comprises an anti-TfRFab covalently linked via a lysine to the 5′ end of an oligonucleotide,wherein the anti-TfR Fab comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 102 and a light chain comprising the aminoacid sequence of SEQ ID NO: 95; wherein the complex has the structureof:

wherein n is 3 and m is 4.

In some embodiments, in any one of the examples of complexes describedherein, L1 is any one of the spacers described herein.

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide, wherein L2is

In some embodiments, L1 is:

wherein the piperazine moiety links to the oligonucleotide.

In some embodiments, L1 is

In some embodiments, L1 is linked to a 5′ phosphate of theoligonucleotide.

In some embodiments, L1 is optional (e.g., need not be present).

IV. Formulations

The anti-TfR antibodies or complexes provided herein may be formulatedin any suitable manner. Generally, the antibodies or complexes providedherein are formulated in a manner suitable for pharmaceutical use. Forexample, the antibodies or complexes can be delivered to a subject usinga formulation that minimizes degradation, facilitates delivery and/or(e.g., and) uptake, or provides another beneficial property to thecomplexes in the formulation. In some embodiments, provided herein arecompositions comprising the antibodies or complexes and pharmaceuticallyacceptable carriers. Such compositions can be suitably formulated suchthat when administered to a subject, either into the immediateenvironment of a target cell or systemically, a sufficient amount of thecomplexes enter target muscle cells. In some embodiments, antibodies orcomplexes are formulated in buffer solutions such as phosphate-bufferedsaline solutions, liposomes, micellar structures, and capsids.

It should be appreciated that, in some embodiments, compositions mayinclude separately one or more components of complexes provided herein(e.g., anti-TfR antibodies, linkers, molecular payloads, or precursormolecules of any one of them).

In some embodiments, antibodies or complexes are formulated in water orin an aqueous solution (e.g., water with pH adjustments). In someembodiments, antibodies or complexes are formulated in basic bufferedaqueous solutions (e.g., PBS). In some embodiments, formulations asdisclosed herein comprise an excipient. In some embodiments, anexcipient confers to a composition improved stability, improvedabsorption, improved solubility and/or (e.g., and) therapeuticenhancement of the active ingredient. In some embodiments, an excipientis a buffering agent (e.g., sodium citrate, sodium phosphate, a trisbase, or sodium hydroxide) or a vehicle (e.g., a buffered solution,petrolatum, dimethyl sulfoxide, or mineral oil).

In some embodiments, a complex or component thereof (e.g.,oligonucleotide or antibody) is lyophilized for extending its shelf-lifeand then made into a solution before use (e.g., administration to asubject). Accordingly, an excipient in a composition comprising acomplex, or component thereof, described herein may be a lyoprotectant(e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone),or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

In some embodiments, a pharmaceutical composition is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, administration. Typically, the route of administration isintravenous or subcutaneous.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (for example,glycerol, propylene glycol, and liquid polyethylene glycol, and thelike), and suitable mixtures thereof. In some embodiments, formulationsinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride in the composition. Sterileinjectable solutions can be prepared by incorporating the complexes in arequired amount in a selected solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization.

In some embodiments, a composition may contain at least about 0.1% ofthe complex, or component thereof, or more, although the percentage ofthe active ingredient(s) may be between about 1% and about 80% or moreof the weight or volume of the total composition. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

V. Methods of Use

Some aspects of the present disclosure provide various uses of theanti-TfR antibodies, antibody fragments or variants, nucleic acidsencoding such, and complexes described herein, including in research,diagnostic methods, detection methods, and therapeutic methods. In someembodiments, the anti-TfR antibodies described herein is used fordelivering a molecular payload (e.g., a diagnostic or therapeutic agent)to a target cell or tissue that expresses a transferrin receptor. Insome embodiments, the target cell is a muscle cell. In some embodiments,the target tissue is muscle. In some embodiments, the target tissue isbrain. For delivering the molecular payload, the anti-TfR antibody maybe conjugated (e.g., covalently conjugated) to the molecular payload toform a complex.

a. Diagnostic and Detection Methods

Also provided herein are the use of any one of the above describedantibodies, antigen-binding fragments, polynucleotides, vectors or cellsand optionally suitable means in diagnostic and/or (e.g., and) detectionmethods. The antibodies or antigen-binding fragments are, for example,suited for use in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. Examples of immunoassays whichcan utilize the antibody or antigen-binding fragments are competitiveand non-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the Enzyme Linked Immunoassay (ELISA),radioimmunoassay (RIA), the sandwich (immunometric assay), flowcytometry, the western blot assay, immunoprecipitation assays,immunohistochemistry, immuno-microscopy, lateral flowimmuno-chromatographic assays, and proteomics arrays. The antigens andantibodies or antigen-binding fragments can be bound to many differentsolid supports (e.g., carriers, membrane, columns, proteomics array,etc.). Examples of well known solid support materials include glass,polystyrene, polyvinyl chloride, polyvinylidene difluoride,polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses,natural and modified celluloses, such as nitrocellulose,polyacrylamides, agaroses, and magnetite. The nature of the support canbe either fixed or suspended in a solution (e.g., beads).

In some embodiments, any one of the anti-TfR antibodies provided hereinis useful for detecting the presence of transferrin receptor in abiological sample. The term “detecting” as used herein encompassesquantitative or qualitative detection. In certain embodiments, abiological sample comprises a cell or tissue, such as blood, CSF, andBBB-containing tissue. The biological sample can be in vitro (e.g.,cultured) or in vivo (e.g., in a subject). The present disclosure alsocontemplates the use of any one of the anti-TfR antibodies describedherein in research use (e.g., as a reagent for immuno assays such aswestern blotting, immunostaining, ELISA, and/or (e.g., and) FACS).

In some embodiments, an anti-TfR antibody for use in a method ofdiagnosis or detection is provided. In some aspects, a method ofdetecting the presence of transferrin receptor in a biological sample isprovided. In certain embodiments, the method comprises contacting thebiological sample with an anti-TfR antibody as described herein underconditions permissive for binding of the anti-TfR antibody to thetransferrin receptor, and detecting whether a complex is formed betweenthe anti-TfR antibody and the transferrin receptor. Such method may bean in vitro or in vivo method. In some embodiments, an anti-TfR antibodyis used to select subjects eligible for therapy with an anti-TfRantibody, e.g. where transferrin receptor is a biomarker for selectionof patients.

Exemplary disorders that may be diagnosed using an anti-TfR antibodydescribed herein include disorders involving immature red blood cells,due to the fact that transferrin receptor is expressed in reticulocytesand is therefore detectable by any of the antibodies of the invention.Such disorders include anemia and other disorders arising from reducedlevels of reticulocytes, or congenital polycythemia or neoplasticpolycythemia vera, where raised red blood cell counts due tohyperproliferation of, e.g., reticulocytes, results in thickening ofblood and concomitant physiological symptoms.

In some embodiments, to detect the presence/level of transferrinreceptor in a biological sample, labeled anti-TfR antibodies are used.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes 32P, 14C, 125I, 3H, and 131,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like. In someembodiments, the detectable label is an agent suitable for detectingtransferrin receptor in a cell in vitro, which can be a radioactivemolecule, a radiopharmaceutical, or an iron oxide particle. Radioactivemolecules suitable for in vivo imaging include, but are not limited to,¹²²I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁸F, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ²¹¹At, ²²⁵Ac,¹⁷⁷Lu, ¹⁵³Sm, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, ²¹³Bi, ²¹²Bi, ²¹²Pb, and ⁶⁷Ga.Exemplary radiopharmaceuticals suitable for in vivo imaging include¹¹¹In Oxyquinoline, ¹³¹I Sodium iodide, ⁹⁹mTc Mebrofenin, and ⁹⁹mTc RedBlood Cells, ¹²³I Sodium iodide, ⁹⁹mTc Exametazime, ⁹⁹mTc MacroaggregateAlbumin, ⁹⁹mTc Medronate, ⁹⁹mTc Mertiatide, ⁹⁹mTc Oxidronate, ⁹⁹mTcPentetate, ⁹⁹mTc Pertechnetate, ⁹⁹mTc Sestamibi, ⁹⁹MTC Sulfur Colloid,⁹⁹mTc Tetrofosmin, Thallium-201, or Xenon-133.

In certain embodiments, the anti-TfR antibody described herein can beused to deliver a detectable label to a target cell or tissue (e.g.,muscle cell or across the blood brain barrier to the brain) forvisualization of the cell or tissue (e.g., by fluorescent microscopy orby magnetic resonance imaging (MRI). Any of the detectable labelsdescribed herein can be used for this purpose.

In some embodiments, the anti-TfR antibody used in a diagnostic ordetection method lacks effector function or has reduced effectorfunction. In some embodiments, the anti-TfR antibody used in adiagnostic/detection method is engineered to have no or reduced effectorfunction (e.g., by using a Fab, modifying the Ig backbone, introducingone or more Fc mutations reducing or eliminating effector function,and/or (e.g., and) modifying the glycosylation state of the antibody).

Various techniques are available for determining binding of the antibodyto the transferrin receptor. One such assay is an enzyme linkedimmunosorbent assay (ELISA) for confirming an ability to bind to humantransferrin receptor (and brain antigen). According to this assay,plates coated with antigen (e.g. recombinant transferrin receptor) areincubated with a sample comprising the anti-TfR antibody and binding ofthe antibody to the antigen of interest is determined.

To perform a diagnostic assay in vivo, a suitable amount of anti-TfRantibodies, conjugated with a label (e.g., an imaging agent or acontrast agent), can be administered to a subject in need of theexamination. Presence of the labeled antibody can be detected based onthe signal released from the label by routine methods. Assays forevaluating uptake of systemically administered antibody and otherbiological activity of the antibody are known to those skilled in theart.

To perform scientific research assays, an anti-TfR antibody can be usedto study bioactivity of transferrin receptor and/or (e.g., and) detectthe presence of transferrin receptor intracellularly. For example, asuitable amount of anti-TfR antibody can be brought in contact with asample (e.g. a new cell type that is not previously identified astransferrin receptor producing cells) suspected of producing transferrinreceptor. The antibody and the sample may be incubated under suitableconditions for a suitable period to allow for binding of the antibody tothe transferrin receptor antigen. Such an interaction can then bedetected via routine methods, e.g., ELISA, histological staining orFACS.

b. Treatment Methods

The anti-TfR antibodies described herein can be used for deliveringmolecular payloads that are therapeutic agents (e.g., oligonucleotides,peptides/proteins, nucleic acid constructs, etc.). In some aspects, thepresent disclosure also provides complexes comprising the anti-TfRantibodies covalently linked to a molecular payload for use in treatingdiseases.

In some aspects, complexes comprising an anti-TfR antibody covalentlylinked to a molecular payload as described herein are effective intreating a muscle disease (e.g., a rare muscle disease or muscleatrophy). In some embodiments, complexes are effective in treating arare muscle disease provided in Table 6. In some embodiments, a muscledisease is associated with a disease allele, for example, a diseaseallele for a particular muscle disease may comprise a genetic alterationin a corresponding gene listed in Table 6.

In some embodiments, complexes are effective in treating muscle atrophyassociated with the activity of one or more genes listed in Table 6under the “Muscle Atrophy Gene Targets” section. In some embodiments,muscle atrophy is due to a chronic illness, including AIDS, congestiveheart failure, cancer, chronic obstructive pulmonary disease, and renalfailure, or muscle disuse.

In other aspects, complexes comprising an anti-TfR antibody covalentlylinked to a molecular payload as described herein are effective intreating a neurological disease. In some embodiments, neurologicaldiseases include, but are not limited to, neuropathy, amyloidosis,cancer, an ocular disease or disorder, viral or microbial infection,inflammation, ischemia, neurodegenerative disease, seizure, behavioraldisorders, and a lysosomal storage disease. For the purposes of thisapplication, the CNS will be understood to include the eye, which isnormally sequestered from the rest of the body by the blood-retinabarrier. Specific examples of neurological disorders include, but arenot limited to, neurodegenerative diseases (including, but not limitedto, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome,olivopontocerebellar atrophy, Parkinson's disease, multiple systematrophy, striatonigral degeneration, tauopathies (including, but notlimited to, Alzheimer disease and supranuclear palsy), prion diseases(including, but not limited to, bovine spongiform encephalopathy,scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), bulbar palsy, motor neuron disease, andnervous system heterodegenerative disorders (including, but not limitedto, Canavan disease, Huntington's disease, neuronalceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkeskinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome,lafora disease, Rett syndrome, hepatolenticular degeneration,Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia(including, but not limited to, Pick's disease, and spinocerebellarataxia), cancer (e.g. of the CNS, including brain metastases resultingfrom cancer elsewhere in the body). In some embodiments, for treating aneurological disease, the complex comprises an anti-TfR antibodydescribed herein conjugated to a drug for treating a neurologicaldisease (e.g., the drugs listed in Table 7).

In some embodiments, a subject may be a human subject, a non-humanprimate subject, a rodent subject, or any suitable mammalian subject. Insome embodiments, a subject may have a muscle disease provided in Table6. In some embodiments, a subject may have muscle atrophy, or be at riskof developing muscle atrophy.

An aspect of the disclosure includes a method involving administering toa subject an effective amount of a complex as described herein. In someembodiments, an effective amount of a pharmaceutical composition thatcomprises a complex comprising an anti-TfR antibody covalently linked toa molecular payload can be administered to a subject in need oftreatment. In some embodiments, a pharmaceutical composition comprisinga complex as described herein may be administered by a suitable route,which may include intravenous administration, e.g., as a bolus or bycontinuous infusion over a period of time. In some embodiments,intravenous administration may be performed by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intra-articular,intrasynovial, or intrathecal routes. In some embodiments, apharmaceutical composition may be in solid form, aqueous form, or aliquid form. In some embodiments, an aqueous or liquid form may benebulized or lyophilized. In some embodiments, a nebulized orlyophilized form may be reconstituted with an aqueous or liquidsolution.

Compositions for intravenous administration may contain various carrierssuch as vegetable oils, dimethylactamide, dimethyformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols(glycerol, propylene glycol, liquid polyethylene glycol, and the like).For intravenous injection, water soluble antibodies can be administeredby the drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising an anti-TfR antibody covalently linked to a molecularpayload is administered via site-specific or local delivery techniques.Examples of these techniques include implantable depot sources of thecomplex, local delivery catheters, site specific carriers, directinjection, or direct application.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising an anti-TfR antibody covalently linked to a molecularpayload is administered at an effective concentration that conferstherapeutic effect on a subject. Effective amounts vary, as recognizedby those skilled in the art, depending on the severity of the disease,unique characteristics of the subject being treated, e.g. age, physicalconditions, health, or weight, the duration of the treatment, the natureof any concurrent therapies, the route of administration and relatedfactors. These related factors are known to those in the art and may beaddressed with no more than routine experimentation. In someembodiments, an effective concentration is the maximum dose that isconsidered to be safe for the patient. In some embodiments, an effectiveconcentration will be the lowest possible concentration that providesmaximum efficacy.

Empirical considerations, e.g. the half-life of the complex in asubject, generally will contribute to determination of the concentrationof pharmaceutical composition that is used for treatment. The frequencyof administration may be empirically determined and adjusted to maximizethe efficacy of the treatment.

Generally, for administration of any of the complexes described herein,an initial candidate dosage may be about 1 to 100 mg/kg, or more,depending on the factors described above, e.g. safety or efficacy. Insome embodiments, a treatment will be administered once. In someembodiments, a treatment will be administered daily, biweekly, weekly,bimonthly, monthly, or at any time interval that provide maximumefficacy while minimizing safety risks to the subject. Generally, theefficacy and the treatment and safety risks may be monitored throughoutthe course of treatment

The efficacy of treatment may be assessed using any suitable methods. Insome embodiments, the efficacy of treatment may be assessed byevaluation of observation of symptoms associated with a muscle diseaseand/or (e.g., and) muscle atrophy.

In some embodiments, a pharmaceutical composition that comprises acomplex comprising an anti-TfR antibody covalently linked to a molecularpayload described herein is administered to a subject at an effectiveconcentration sufficient to inhibit activity or expression of a targetgene by at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90% or at least95% relative to a control, e.g. baseline level of gene expression priorto treatment.

In some embodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising an anti-TfR antibodycovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1-5, 1-10, 5-15, 10-20, 15-30, 20-40, 25-50, or more days. In someembodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising an anti-TfR antibodycovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In someembodiments, a single dose or administration of a pharmaceuticalcomposition that comprises a complex comprising an anti-TfR antibodycovalently linked to a molecular payload described herein to a subjectis sufficient to inhibit activity or expression of a target gene for atleast 1, 2, 3, 4, 5, or 6 months.

In some embodiments, a pharmaceutical composition may comprise more thanone complex comprising an anti-TfR antibody covalently linked to amolecular payload. In some embodiments, a pharmaceutical composition mayfurther comprise any other suitable therapeutic agent for treatment of asubject, e.g. a human subject having a muscle disease (e.g., a muscledisease provided in Table 6). In some embodiments, the other therapeuticagents may enhance or supplement the effectiveness of the complexesdescribed herein. In some embodiments, the other therapeutic agents mayfunction to treat a different symptom or disease than the complexesdescribed herein.

c. Kits for Therapeutic and Diagnostic Applications

The present disclosure also provides kits for the therapeutic ordiagnostic applications as disclosed herein. Such kits can include oneor more containers comprising an anti-TfR antibody, e.g., any of thosedescribed herein.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise a description of administration of theanti-TfR antibody to treat, delay the onset, or alleviate a targetdisease as those described herein. The kit may further comprise adescription of selecting an individual suitable for treatment based onidentifying whether that individual has the target disease. In stillother embodiments, the instructions comprise a description ofadministering an antibody to an individual at risk of the targetdisease.

The instructions relating to the use of an anti-TfR antibody generallyinclude information as to dosage, dosing schedule, and route ofadministration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used fortreating, delaying the onset and/or (e.g., and) alleviating a disease ordisorder treatable by modulating immune responses, such as autoimmunediseases. Instructions may be provided for practicing any of the methodsdescribed herein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like.

Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer) or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is an anti-TfR antibody as thosedescribed herein.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

Also provided herein are kits for use in detecting transferrin receptorin a sample. Such a kit may comprise any of the anti-TfR antibodiesdescribed herein. In some instances, the anti-TfR antibody can beconjugated with a detectable label as those described herein. As usedherein, “conjugated” or “attached” means two entities are associated,preferably with sufficient affinity that the therapeutic/diagnosticbenefit of the association between the two entities is realized. Theassociation between the two entities can be either direct or via alinker, such as a polymer linker. Conjugated or attached can includecovalent or noncovalent bonding as well as other forms of association,such as entrapment, e.g., of one entity on or within the other, or ofeither or both entities on or within a third entity, such as a micelle.

Alternatively or in addition (e.g., in addition), the kit may comprise asecondary antibody capable of binding to anti-TfR antibody. The kit mayfurther comprise instructions for using the anti-TfR antibody fordetecting transferrin receptor.

EXAMPLES Example 1: Humanized Anti-TfR1 Antibodies

The anti-TfR antibodies shown in Table 2 were subjected to humanizationand mutagenesis to reduce manufacturability liabilities. The humanizedvariants were screened and tested for their binding properties andbiological actives. Humanized variants of anti-TfR1 heavy and lightchain variable regions (5 variants each) were designed using CompositeHuman Technology. Genes encoding Fabs having these heavy and light chainvariable regions were synthesized, and vectors were constructed toexpress each humanized heavy and light chain variant. Subsequently, eachvector was expressed on a small scale and the resultant humanizedanti-TfR1 Fabs were analyzed. Humanized Fabs were selected for furthertesting based upon several criteria including Biacore assays of antibodyaffinity for the target antigen, relative expression, percent homologyto human germline sequence, and the number of MHC class II predicted Tcell epitopes (determined using iTope™ MCH class II in silico analysis).

Potential liabilities were identified within the parental sequence ofsome antibodies by introducing amino acid substitutions in the heavychain and light chain variable regions. These substitutions were chosenbased on relative expression levels, iTope™ score and relative K_(D)from Biacore single cycle kinetics analysis. The humanized variants weretested and variants were selected initially based upon affinity for thetarget antigen. Subsequently, the selected humanized Fabs were furtherscreened based on a series of biophysical assessments of stability andsusceptibility to aggregation and degradation of each analyzed variant,shown in Table 8 and Table 9. The selected Fabs were analyzed for theirproperties binding to TfR1 by kinetic analysis. The results of theseanalyses are shown in Table 10. For conjugates shown in Table 8 andTable 9, the selected humanized Fabs were conjugated to a DMPK-targetingoligonucleotide ASO300. The selected Fabs are thermally stable, asindicated by the comparable binding affinity to human and cyno TfR1after been exposed to high temperature (40° C.) for 9 days, compared tobefore the exposure (see Table 10).

TABLE 8 Biophysical assessment data for humanized anti-TfR Fabs Variant3M12 3M12 3M12 3M12 3A4 (VH3- Criteria (VH3/Vk2) (VH3/Vk3) (VH4/Vk2)(VH4/Vk3) N54T/Vk4) Binding Affinity  395 pM  345 pM  396 pM  341 pM3.09 nM (Biacore d0) Binding Affinity  567 pM  515 pM  510 pM  486 pM3.01 nM (Biacore d25) Fab binding affinity 0.8 nM/9.9 nM 0.6 nM/4.7 nM 0.4 nM/1.4 nM  0.5 nM/2.2 nM  2.6 nM/156 nM* ELISA (human/cyno TfR1)Conjugate binding 2.2 nM/2.9 nM N/A N/A  1.7 nM/2.1 nM  2.8 nM/4.7 nMaffinity ELISA (human/cyno TfR1) . . . Variant 3A4 (VH3- 3A4 5H12 (VH5-5H12 (VH5- 5H12 (VH4- Criteria N54S/Vk4) (VH3/Vk4) C33Y/Vk3) C33D/Vk4)C33Y/Vk4) Binding Affinity 1.34 nM  1.5 nM  627 pM  991 pM  626 pM(Biacore d0) Binding Affinity 1.39 nM 1.35 nM 1.07 nM 3.01 nM 1.33 nM(Biacore d25) Fab binding affinity 1.6 nM/398 nM* 1.5 nM/122 nM*  6.3nM/2.1 nM  6.0 nM/3.5 nM  2.8 nM/3.3 nM ELISA (human/cyno TfR1)Conjugate binding 2.9 nM/7.8 nM 2.8 nM/7.6 nM 33.4 nM/2.3 nM 110 nM/10.2nM 23.7 nM/3.3 nM affinity ELISA (human/cyno TfR1) *Regains cyno bindingafter conjugation;

TABLE 9 Thermal Stability for humanized anti-TfR Fabs and conjugatesVariant 3M12 3M12 3M12 3M12 3A4 (VH3- Criteria (VH3/VK2) (VH3/VK3)(VH4/Vk2) (VH4/Vk3) N54T/Vk4) Binding affinity hTfR1 0.8 0.6 0.4 0.5 2.6d0 (nM) Binding affinity hTfRI 0.98 1.49 0.50 0.28 0.40 d9 (nM) Bindingaffinity cyno 9.9 4.7 1.4 2.2 156 TfR1 d0 (nM) Binding affinity cyno19.51 15.58 5.01 16.40 127.50 TfR1 d9 (nM) DMPK oligo conjugate 1.14 N/AN/A 1.18 2.22 binding to hTfR1 (nM) DMPK oligo conjugate 2.26 N/A N/A1.85 5.12 binding to cyno TfRl (nM) . . . Variant 3A4 (VH3- 3A4 5H12(VH5- 5H12 (VH5- 5H12 (VH4- Criteria N54S/Vk4) (VH3/Vk4) C33Y/Vk3)C33D/Vk4) C33Y/Vk4) Binding affinity hTfR1 1.6 1.5 6.3 6 2.8 d0 (nM)Binding affinity hTfR1 0.65 0.46 71.90 92.34 1731.00 d9 (nM) Bindingaffinity cyno 398 122 2.1 3.5 3.3 TfR1 d0 (nM) Binding affinity cyno248.30 878.40 0.69 0.63 0.26 TfR1 d9 (nM) DMPK oligo conjugate 2.712.837 N/A 110.5 13.9 binding to hTfR1 (nM) DMPK oligo conjugate 4.17.594 N/A 10.18 13.9 binding to cyno TfR1 (nM)

TABLE 10 Kinetic analysis of humanized anti-TfR. Fabs binding to TfR1Humanized anti- TfR Fabs k_(a) (1/Ms) k_(d) (1/s) K_(D) (M) R_(MAX) Chi²(RU²) 3A4 (VH3/Vk4) 7.65E+10 1.15E+02 1.50E−09 48.0 0.776 3A4(VH3-N54S/Vk4) 4.90E+10 6.56E+01 1.34E−09 49.4 0.622 3A4 (VH3-N54T/Vk4)2.28E+05 7.05E−04 3.09E−09 61.1 1.650 3M12 (VH3/Vk2) 2.64E+05 1.04E−043.95E−10 78.4 0.037 3M12 (VH3/Vk3) 2.42E+05 8.34E−05 3.45E−10 91.1 0.0253M12 (VH4/Vk2) 2.52E+05 9.98E−05 3.96E−10 74.8 0.024 3M12 (VH4/Vk3)2.52E+05 8.61E−05 3.41E−10 82.4 0.030 5H12 (VH5-C33D/Vk4) 6.78E+056.72E−04 9.91E−10 49.3 0.093 5H12 (VH5-C33Y/Vk3) 1.95E+05 1.22E−046.27E−10 68.5 0.021 5H12 (VH5-C33Y/Vk4) 1.86E+05 1.17E−04 6.26E−10 75.20.026Binding of Humanized Anti-TfR1 Fabs to TfR1 (ELISA)

To measure binding of humanized anti-TfR antibodies to TfR1, ELISAs wereconducted. High binding, black, flat bottom, 96 well plates (Corning#3925) were first coated with 100 μL/well of recombinant huTfR1 at 1μg/mL in PBS and incubated at 4° C. overnight. Wells were emptied andresidual liquid was removed. Blocking was conducted by adding 200 μL of1% BSA (w/w) in PBS to each well. Blocking was allowed to proceed for 2hours at room temperature on a shaker at 300 rpm. After blocking, liquidwas removed and wells were washed three times with 300 μL of TBST.Anti-TfR1 antibodies were then added in 0.5% BSA/TBST in triplicate inan 8 point serial dilution (dilution range 5 μg/mL-5 ng/mL). A positivecontrol and isotype controls were also included on the ELISA plate. Theplate was incubated at room temperature on an orbital shaker for 60minutes at 300 rpm, and the plate was washed three times with 300 μL ofTBST. Anti-(H+L)IgG-A488 (1:500) (Invitrogen #A11013) was diluted in0.5% BSA in TBST, and 100 μL was added to each well. The plate was thenallowed to incubate at room temperature for 60 minutes at 300 rpm onorbital shaker. The liquid was removed and the plate was washed fourtimes with 3004 of TBST. Absorbance was then measured at 495 nmexcitation and 50 nm emission (with a 15 nm bandwidth) on a platereader. Data was recorded and analyzed for EC₅₀. The data for binding tohuman TfR1 (hTfR1) for the humanized 3M12, 3A4 and 5H12 Fabs are shownin FIGS. 1A, 1C, and 1E, respectively. ELISA measurements were conductedusing cynomolgus monkey (Macaca fascicularis) TfR1 (cTfR1) according tothe same protocol described above for hTfR1, and results are shown inFIGS. 1B, 1D, and 1F.

Results of these two sets of ELISA analyses for binding of the humanizedanti-TfR Fabs to hTfR1 and cTfR1 demonstrate that humanized 3M12 Fabsshow consistent binding to both hTfR1 and cTfR1, and that humanized 3A4Fabs show decreased binding to cTfR1 relative to hTfR1.

Antibody-oligonucleotide conjugates were prepared using six humanizedanti-TfR Fabs, each of which were conjugated to a DMPK targetingoligonucleotide A50300. Conjugation efficiency and down-streampurification were characterized, and various properties of the productconjugates were measured. The results demonstrate that conjugationefficiency was robust across all 10 variants tested, and that thepurification process (hydrophobic interaction chromatography followed byhydroxyapatite resin chromatography) were effective. The purifiedconjugates showed a >97% purity as analyzed by size exclusionchromatography.

Several humanized Fabs were tested in cellular uptake experiments toevaluate TfR1-mediated internalization. To measure such cellular uptakemediated by antibodies, humanized anti-TfR Fab conjugates were labeledwith Cypher5e, a pH-sensitive dye. Rhabdomyosarcoma (RD) cells weretreated for 4 hours with 100 nM of the conjugates, trypsinized, washedtwice, and analyzed by flow cytometry. Mean Cypher5e fluorescence(representing uptake) was calculated using Attune NxT software. As shownin FIG. 2 , the humanized anti-TfR Fabs show similar or greaterendosomal uptake compared to a positive control anti-TfR1 Fab. Similarinternalization efficiencies were observed for different oligonucleotidepayloads. An anti-mouse UR antibody was used as the negative control.Cold (non-internalizing) conditions abrogated the fluorescence signal ofthe positive control antibody-conjugate (data not shown), indicatingthat the positive signal in the positive control and humanized anti-TfRFab-conjugates is due to internalization of the Fab-conjugates.

Conjugates of six humanized anti-TfR Fabs of were also tested forbinding to hTfR1 and cTfR1 by ELISA, and compared to the unconjugatedforms of the humanized Fabs. Results demonstrate that humanized 3M12 and5H12 Fabs maintain similar levels of hTfR1 and cTfR1 binding afterconjugation relative to their unconjugated forms (3M12, FIGS. 3A and 3B;5H12, FIGS. 3E and 3F). Interestingly, 3A4 clones show improved bindingto cTfR1 after conjugation relative to their unconjugated forms (FIGS.3C and 3D).

As used in this Example, the term ‘unconjugated’ indicates that theantibody was not conjugated to an oligonucleotide.

Example 2. Knockdown of DMPK mRNA Level Facilitated byAntibody-Oligonucleotide Conjugates In Vitro

Conjugates containing humanized anti-TfR Fabs 3M12(VH3/Vk2), 3M-12(VH4/Vk3), and 3A4(VH3-N54S/Vk4) were conjugated to a DMPK-targetingantisense oligonucleotide ASO300 and were tested in rhabdomyosarcoma(RD) cells for knockdown of DMPK transcript expression. Antibodies wereconjugated to ASO300 via the linker shown in Formula (C).

RD cells were cultured in a growth medium of DMEM with glutamine,supplemented with 10% FBS and penicillin/streptomycin until nearlyconfluent. Cells were then seeded into a 96 well plate at 20K cells perwell and were allowed to recover for 24 hours. Cells were then treatedwith the conjugates for 3 days. Total RNA was collected from cells, cDNAwas synthesized and DMPK expression was measured by qPCR.

Results in FIG. 4 show that DMPK expression level was reduced in cellstreated with each indicated conjugate, relative to expression inPBS-treated cells, indicating that the humanized anti-TfR Fabs are ableto mediate the uptake of the DMPK-targeting oligonucleotide by the RDcells and that the internalized DMPK-targeting oligonucleotide areeffective in knocking down DMPK mRNA level.

Example 3. Serum Stability of the Linker Linking the Anti-TfR Antibodyand the Molecular Payload

Oligonucleotides which were linked to antibodies in examples wereconjugated via a cleavable linker shown in Formula (C). It is importantthat the linker maintain stability in serum and provide release kineticsthat favor sufficient payload accumulation in the targeted muscle cell.This serum stability is important for systemic intravenousadministration, stability of the conjugated oligonucleotide in thebloodstream, delivery to muscle tissue and internalization of thetherapeutic payload in the muscle cells. The linker has been confirmedto facilitate precise conjugation of multiple types of payloads(including ASOs, siRNAs and PMOs) to Fabs. This flexibility enabledrational selection of the appropriate type of payload to address thegenetic basis of each muscle disease. Additionally, the linker andconjugation chemistry allowed the optimization of the ratio of payloadmolecules attached to each Fab for each type of payload, and enabledrapid design, production and screening of molecules to enable use invarious muscle disease applications.

FIG. 5 shows serum stability of the linker in vivo, which was comparableacross multiple species over the course of 72 hours after intravenousdosing. At least 75% stability was measured in each case at 72 hoursafter dosing.

Example 4. Exon-Skipping Activity of Anti-TfR Conjugates in DMD PatientMyotubes

In this study, the exon-skipping activities of anti-TfR conjugatescontaining an anti-TfR Fab (3M12 VH3/VK2, 3M12 VH4/VK3, and 3A4 VH3N54S/VK4) conjugated to a DMD exon51-skipping oligonucleotide wereevaluated. Immortalized human myoblasts bearing an exon 52 deletion werethawed and seeded at a density of 1e6 cell/flask in Promocell SkeletalCell Growth Media (with 5% FBS and 1× Pen-Strep) and allowed to grow toconfluency. Once confluent, cells were trypsinized and pelleted viacentrifugation and resuspended in fresh Promocell Skeletal Cell GrowthMedia. The cell number was counted and cells were seeded intoMatrigel-coated 96-well plates at a density of 50 k cells/well. Cellswere allowed to recover for 24 hours. Cells were induced todifferentiate by aspirating the growth media and replacing withdifferentiation media with no serum. Cells were then treated withconjugated or unconjugated DMD exon skipping oligonucleotide at 10 μM.Cells were incubated with test articles for ten days then total RNA washarvested from the 96 well plates. cDNA synthesis was performed on 75 ngof total RNA, and mutation specific PCRs were performed to evaluate thedegree of exon 51 skipping in each cell type. Mutation-specific PCRproducts were run on a 4% agarose gel and visualized using SYBR gold.Densitometry was used to calculate the relative amounts of the skippedand unskipped amplicon and exon skipping was determined as a ratio ofthe exon 51 skipped amplicon divided by the total amount of ampliconpresent:

${\%{Exon}{Skipping}} = {\frac{{Skipped}{Amplicon}}{\left( {{{Skipped}{Amplicon}} + {{Unskipped}{Amplicon}}} \right)}*100}$

The data demonstrates that the conjugates with either 3M12 VH3/Vκ2 or3M12 VH4/Vκ3 Fab conjugated to the DMD exon 51-skipping oligonucleotideresulted in enhanced exon skipping compared to the unconjugated DMD exonskipping oligonucleotide in patient myotubes (FIG. 6 ).

As used in this Example, the term ‘unconjugated’ indicates that theoligonucleotide was not conjugated to an antibody.

Example 5. In Vivo Activity of Anti-TfR Conjugates in hTfR1 Mice

In DM1, the higher than normal number of CUG repeats form large hairpinloops that remain trapped in the nucleus, forming nuclear foci that bindsplicing proteins and inhibit the ability of splicing proteins toperform their normal function. When toxic nuclear DMPK levels arereduced, the nuclear foci are diminished, releasing splicing proteins,allowing restoration of normal mRNA processing, and potentially stoppingor reversing disease progression.

The in vivo activity of conjugates containing an anti-TfR Fab (control,3M12 VH3/VK2, 3M12 VH4/VK3, 3A4 VH3 N54S/VK4) conjugated to theDMPK-targeting oligonucleotide A50300 in reducing DMPK mRNA level inmultiple muscle tissues following systemic intravenous administration inmice was evaluated.

Male and female C57BL/6 mice where one TfR1 allele was replaced with ahuman TFR1 allele were administered between the ages of 5 and 15 weeksaccording to the dosing schedule outlined in Table 11 and in FIG. 7A.Mice were sacrificed 14 days after the first injection and selectedmuscles collected as indicated in Table 12.

TABLE 11 Ani- Treat- Treat- Dose Dose Dosing Terminal mal ment mentLevel Volume Regi- Time Group No. Antibody Oligo (mg/kg) (mL/kg) menPoint 1 4 Vehicle NA 0 10 Day 0 Day 14 2 4 NA ASO300 10 5.0 and 3 4control ASO300 10.2 Day anti-TfR 7 by Fab IV 4 4 3M12 ASO300 11.5 VH3/VK2 5 4 3M12 ASO300 10.1 VH4/ VK3 6 4 3A4 VH3 ASO300 10.7 N54S/ VK4

TABLE 12 Tissue Storage Gastrocnemius Right leg of each animal stored inRNALater at −80° C. Tibialis One leg (R) of each animal stored Anteriorin RNALater at −80° C. Heart Dissect transversally and store the apex inRNAlater at −80° C. Diaphragm Split in half and collect one half inRNAlater at −80° C.

Total RNA was extracted on a Maxwell Rapid Sample Concentrator (RSC)Instrument using kits provided by the manufacturer (Promega). PurifiedRNA was reverse-transcribed and levels of Dmpk and Ppib transcriptsdetermined by qRT-PCR with specific TaqMan assays (ThermoFIsher). Logfold changes in Dmpk expression were calculated according to the2^(−ΔΔCT) method using Ppib as the reference gene and mice injected withvehicle as the control group. Statistical significance in differences ofDmpk expression between control mice and mice administered with theconjugates were determined by one-way ANOVA with Dunnet's correction formultiple comparisons. As shown in FIGS. 7B-7E, the tested conjugatesshowed robust activity in reducing DMPK mRNA level in vivo in variousmuscle tissues.

Example 6. Epitope Mapping

In order to determine the epitope of the hTfR1/anti-TfR Fab (3M12VH4/Vk3) complex with high resolution, the protein complex was incubatedwith deuterated cross-linkers and subjected to multi-enzymatic cleavage.After enrichment of the cross-linked peptides, the samples were analyzedby high resolution mass spectrometry (nLC-LTQ-Orbitrap MS) and the datagenerated were analyzed using XQuest and Stavrox software.

20 μL of the hTfR1 (the extracellular domain of human TfR1 as set forthin SEQ ID NO: 35, amino acids C89-F760)/anti-TfR mixture prepared wasmixed with 2 μL of DSS d0/d12 (2 mg/mL; DMF) before 180 minutesincubation time at room temperature. After incubation, reaction wasstopped by adding 1 μL of Ammonium Bicarbonate (20 mM finalconcentration) before 1 hour incubation time at room temperature. Then,the solution was dried using a speedvac before H₂O 8M urea suspension(20 μL). After mixing, 2 μl of DTT (500 mM) were added to the solution.The mixture was then incubated 1 hour at 37° C. After incubation, 2 μlof iodoacetamide (1M) were added before 1 hour incubation time at roomtemperature, in a dark room. After incubation, 80 μl of the proteolyticbuffer were added. The trypsin buffer contains 50 mM Ambic pH 8.5, 5%acetonitrile; The Chymotrypsin buffer contains Tris HCl 100 mM, CaCl₂ 10mM pH 7.8; The ASP-N buffer contains Phopshate buffer 50MM pH 7.8; Theelastase buffer contains Tris HCl 50 mM pH 8.0 and the thermolysinbuffer contains Tris HCl 50 mM, CaCl₂) 0.5 mM pH 9.0.

100 μl of the reduced/alkyled hTfR1/anti-TfR Fab mixture was mixed with4 μl of trypsin (Promega) with the ratio 1/100. The proteolytic mixturewas incubated overnight at 37° C.

100 μl of the reduced/alkyled hTfR1/anti-TfR Fab mixture was mixed with2 μl of chymotrypsin (Promega) with the ratio 1/200. The proteolyticmixture was incubated overnight at 25° C.

100 μl of the reduced/alkyled hTfR1/anti-TfR Fab mixture was mixed with2 μl of ASP-N(Promega) with the ratio 1/200. The proteolytic mixture wasincubated overnight at 37° C.

100 μl of the reduced/alkyled hTfR1/anti-TfR Fab mixture was mixed with4 μl of elastase (Promega) with the ratio 1/100. The proteolytic mixturewas incubated overnight at 37° C.

100 μl of the reduced/alkyled hTfR1/anti-TfR Fab mixture was mixed with8 μl of thermolysin (Promega) with a ratio 1/50. The proteolytic mixturewas incubated overnight at 70° C. After digestion formic acid 1% finalwas added to the solution.

The samples were analyzed using nLC chromatography in combination withLTQ-Orbitrap mass spectrometry have been used. The cross-linked peptideswere analyzed using Xquest version 2.0 and Stavrox 3.6. software. ThenLC-orbitrap MS/MS analysis detected 15 cross-linked peptides betweenhTfr1 and the anti-TfR Fab. The analysis indicates that the interactionincludes the following amino acids on hTfR1: K261, 5273, Y282, T362,S368, 5370, and K371 of SEQ ID NO: 105.

Example 7. Characterization of Binding Activities of Anti-TfR Fab 3M12VH4/Vk3

In vitro studies were performed to test the specificity of anti-TfR Fab3M12 VH4/Vk3 for human and cynomolgus monkey TfR1 binding and to confirmits selectivity for human TfR1 vs TfR2. The binding affinity of anti-TfRFab 3M12 VH4/Vk3 to TfR1 from various species was determined using anenzyme-linked immunosorbent assay (ELISA). Serial dilutions of the Fabwere added to plates precoated with recombinant human, cynomolgusmonkey, mouse, or rat TfR1. After a short incubation, binding of the Fabwas quantified by addition of a fluorescently tagged anti-(H+L) IgGsecondary antibody and measurement of fluorescence intensity at 495 nmexcitation and 520 nm emission. The Fab showed strong binding affinityto human and cynomolgus monkey TfR1, and no detectable binding of mouseor rat TfR1 was observed (FIG. 8 ). Surface plasmon resonance (SPR)measurements were also conducted, and results are shown in Table 13. TheK_(d) of the Fab against the human TfR1 receptor was calculated to be7.68×10⁻¹⁰ M and against the cynomolgus monkey TfR1 receptor wascalculated to be 5.18×10⁻⁹M.

TABLE 13 Kinetic analysis of anti-TfR Fab 3M12 VH4/Vk3 binding to humanand cynomolgus monkey TfR1 or human TfR2, measured using surface plasmonresonance Anti-TfR Fab 3M12 VH4/Vk3 k_(a) Target K_(d) (M) (M⁻¹ s⁻¹)k_(d) (s⁻¹) R_(max) R_(es) SD Human TfR1 7.68E−10 1.66E+05 1.27E−041.11E+02 3.45E+00 Cyno TfR1 5.18E−09 9.19E+04 4.76E−04 1.87E+02 6.24E+00Human TfR2 ND ND ND ND ND ND = No detectable binding by SPR (10 pM-100uM)

To test for cross-reactivity of anti-TfR Fab 3M12 VH4/Vk3 to human TfR2,an ELISA was performed. Recombinant human TfR2 protein was platedovernight at 2 μg/mL and was blocked for 1 hour with 1% bovine serumalbumin (BSA) in PBS. Serial dilutions of the Fab or a positive controlanti-TfR2 antibody were added in 0.5% BSA/TBST for 1 hour. Afterwashing, anti-(H+L) IgG-A488 (Invitrogen #MA5-25932) fluorescentsecondary antibody was added at a 1:500 dilution in 0.5% BSA/TBST andthe plate was incubated for 1 hour. Relative fluorescence was measuredusing a Biotek Synergy plate reader at 495 nm excitation and 520 nmemission. No binding of anti-TfR Fab 3M12 VH4/Vk3 to hTfR2 was observed(FIG. 9 ).

Example 8. Serum Stability of Anti-TfR Fab-ASO Conjugate

Anti-TfR Fab VH4/Vk3 was conjugated to a control antisenseoligonucleotide (ASO) via a linker as shown in Formula (C) and theresulting conjugate was tested for stability of the linker conjugatingthe Fab to the ASO. Serum stability was measured by incubatingfluorescently labeled conjugate in PBS or in rat, mouse, cynomolgusmonkey, or human serum and measuring relative fluorescence intensityover time, with higher fluorescence indicating more conjugate remainingintact. FIG. 10 shows serum stability was similar across multiplespecies and remained high after 72 hours.

Example 9. Exon Skipping Activity of Anti-TfR Fab-ASO Conjugate In Vivoin Cynomolgus Monkeys

Anti-TfR Fab 3M12 VH4/Vk3 was conjugated to a dystrophin (DMD)exon51-skipping antisense oligonucleotide (ASO) targeting an exonicsplicing enhancer (ESE) sequence in DMD exon 51. The exon51 skippingoligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) of 30nucleotides in length. The exon skipping activity of the conjugate wastested in vivo in healthy non-human primates. Naïve male cynomolgusmonkeys (n=4-5 per group) were administered two doses of vehicle, 30mg/kg ASO alone, or 122 mg/kg conjugate (30 mg/kg ASO equivalent) viaintravenous infusion on days 1 and 8. Animals were sacrificed andtissues harvested either 2 weeks or 4 weeks after the first dose wasadministered. Total RNA was collected from tissue samples using aPromega Maxwell® RSC instrument and cDNA synthesis was performed usingqScript cDNA SuperMix. Assessment of exon 51 skipping was performedusing end-point PCR.

Capillary electrophoresis of the PCR products was used to assess exonskipping, and % exon 51 skipping was calculated using the followingformula:

${\%{Exon}{Skipping}} = {\frac{{Molarity}{of}{Skipped}{Band}}{{{Molarity}{of}{Skipped}{Band}} + {{Molarity}{of}{Unskipped}{Band}}}*100.}$

TABLE 14 Exon 51 skipping of dystrophin in cynomolgus monkey dystrophinTime 2 weeks 4 weeks ASO ASO Group Vehicle alone^(a) Conjugate alone^(a)Conjugate Conjugate dose^(b) 0   n/a 122     n/a 122     ASO aloneDose^(c) 0   30    30    30    30    Quadriceps ^(d) 0.00 1.216 4.9060.840 1.708 (0.00) (1.083) (3.131) (1.169) (1.395) Diaphragm ^(d) 0.001.891 7.315 0.717 9.225 (0.00) (2.911) (1.532) (1.315) (4.696) Heart^(d) 0.00 0.043 3.42  0.00  4.525 (0.00) (0.096) (1.192) (0.00)  (1.400)Biceps ^(d) 0.00 0.607 3.129 1.214 4.863 (0.00) (0.615) (0.912) (1.441)(3.881) Tibialis anterior ^(d) 0.00 0.699 1.042 0.384 0.816 (0.00)(0.997) (0.685) (0.615) (0.915) Gastrocnemius ^(d) 0.00 0.388 2.4240.00  5.393 (0.00) (0.573) (2.329) (0.00)  (2.695) ^(a)ASO = antisenseoligonucleotide, ^(b)Conjugate doses are listed as mg/kg of anti-TfR Fab3M12 VH4/Vk3-ASO conjugate. ^(c)ASO doses are listed as mg/kg ASOequivalent of the anti-TfR Fab 3M12 VH4/Vk3-ASO dose. ^(d) Exon skippingvalues are mean % exon 51 skipping with standard deviations (n = 5) inparentheses.

Tissue ASO accumulation was also quantified using a hybridization ELISAwith a probe complementary to the ASO sequence. A standard curve wasgenerated and ASO levels (in ng/g) were derived from a linear regressionof the standard curve. The ASO was distributed to all tissues evaluatedat a higher level following the administration of the anti-TfR FabVH4/Vk3-ASO conjugate as compared to the administration of unconjugatedASO. Intravenous administration of unconjugated ASO resulted in levelsof ASO that were close to background levels in all tissues evaluated at2 and 4 weeks after the first does was administered. Administration ofanti-TfR Fab VH4/Vk3-ASO conjugate resulted in distribution of ASOthrough the tissues evaluated with a rank order ofheart>diaphragm>bicep>quadriceps>gastrocnemious>tibialis anterior 2weeks after first dosing. The duration of tissue concentration was alsoassessed. Concentrations of the ASO in quadriceps, bicep and diaphragmdecreased by less than 50% over the time period evaluated (2 to 4weeks), while levels of ASO in the heart, tibialis anterior, andgastrocnemius remained virtually unchanged (Table 15).

As used in this Example, the term ‘unconjugated’ indicates that theoligonucleotide was not conjugated to an antibody.

TABLE 15 Tissue distribution of DMD exon51 skipping ASO in cynomolgusmonkeys Time 2 weeks 4 weeks ASO ASO Group Vehicle alone^(a) Conjugatealone^(a) Conjugate Conjugate Dose^(b) 0   n/a  122 n/a 122 ASO aloneDose^(c) 0   30    30  30  30 Quadriceps ^(d) 0   696.8  2436 197 682(59.05) (868.15)   (954.0) (134) (281) Diaphragm ^(d) 0± 580.02 6750  603131  (144.3)  (360.11) (2256) (120) (1618)  Heart ^(d) 0   1449   27138  943 30410  (396.03)  (1337)    (6315) (1803)  (9247)  Biceps ^(d)0   615.63 2840 130 1326  (69.58) (335.17)    (980.31) (80) (623)Tibialis anterior ^(d) 0   564.71 1591 169 1087  (76.31) (327.88)   (253.50) (110) (514) Gastrocnemius ^(d) 0   705.47 2096 170 1265 (41.15) (863.75)    (474.04)  (69) (272) ^(a)ASO = Antisenseoligonucleotide. ^(b)Conjugate doses are listed as mg/kg of anti-TfR Fab3M12 VH4/Vk3 ASO conjugate. ^(c)ASO doses are listed as mg/kg ASO or ASOequivalent of the anti-TfR Fab 3M12 VH4/Vk3-ASO conjugate dose. ^(d) ASOvalues are mean concentrations of ASO in tissue as ng/g with standarddeviations (n = 5) in parentheses.

Additional Embodiments

1. A humanized antibody that binds to human transferrin receptor (TfR),wherein the antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 69; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 70;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 75;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 75;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 80.

2. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 69 and aVL comprising the amino acid sequence of SEQ ID NO: 70.

3. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 71 and aVL comprising the amino acid sequence of SEQ ID NO: 70.

4. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 72 and aVL comprising an amino acid of SEQ ID NO: 70.

5. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and aVL comprising the amino acid sequence of SEQ ID NO: 74.

6. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 73 and aVL comprising the amino acid sequence of SEQ ID NO: 75.

7. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and aVL comprising the amino acid sequence of SEQ ID NO: 74.

8. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and aVL comprising the amino acid sequence of SEQ ID NO: 75.

9. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and aVL comprising the amino acid sequence of SEQ ID NO: 78.

10. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 79 and aVL comprising the amino acid sequence of SEQ ID NO: 80.

11. The humanized antibody of embodiment 1, wherein the antibodycomprises a VH comprising the amino acid sequence of SEQ ID NO: 77 and aVL comprising the amino acid sequence of SEQ ID NO: 80.

12. The humanized antibody of any one of embodiments 1-11, wherein theantibody is selected from the group consisting of a full-length IgG, aFab fragment, a Fab′ fragment, a F(ab′)2 fragment, an scFv, and an Fv.

13. The humanized antibody of embodiment 12, wherein the antibody is afull-length IgG.

14. The humanized antibody of embodiment 13, wherein the antibodycomprises a heavy chain constant region of the isotype IgG1, IgG2, IgG3,or IgG4.

15. The humanized antibody of embodiment 13 or embodiment 14, whereinthe antibody comprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 84; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 86; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 87; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 88; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 88; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 91; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 91; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 92; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 94; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 92; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

16. The humanized antibody of embodiment 12, wherein the antibody is aFab fragment.

17. The humanized antibody of embodiment 16, wherein the antibodycomprises:

(i) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 97; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(ii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 98; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 99; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 85;

(iv) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(v) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 100; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(vi) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 89;

(vii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 101; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 90;

(viii) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 93;

(ix) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 103; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95; or

(x) a heavy chain comprising an amino acid sequence at least 85%identical to SEQ ID NO: 102; and/or a light chain comprising an aminoacid sequence at least 85% identical to SEQ ID NO: 95.

18. The humanized antibody of embodiment 17, wherein the antibodycomprises:

(i) a heavy chain comprising the amino acid sequence of SEQ ID NO: 97;and/or a light chain comprising the amino acid sequence of SEQ ID NO:85;

(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 98;and/or a light chain comprising the amino acid sequence of SEQ ID NO:85;

(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO: 99;and/or a light chain comprising the amino acid sequence of SEQ ID NO:85;

(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and/or a light chain comprising the amino acid sequence of SEQ ID NO:89;

(v) a heavy chain comprising the amino acid sequence of SEQ ID NO: 100;and/or a light chain comprising the amino acid sequence of SEQ ID NO:90;

(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO: 101;and/or a light chain comprising the amino acid sequence of SEQ ID NO:89;

(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO:101; and/or a light chain comprising the amino acid sequence of SEQ IDNO: 90;

(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO:102; and/or a light chain comprising the amino acid sequence of SEQ IDNO: 93;

(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO: 103;and/or a light chain comprising the amino acid sequence of SEQ ID NO:95; or

(x) a heavy chain comprising the amino acid sequence of SEQ ID NO: 102;and/or a light chain comprising the amino acid sequence of SEQ ID NO:95.

19. The humanized antibody of any one of embodiments 1 to 18, whereinthe equilibrium dissociation constant (K_(D)) of binding of the antibodyto the transferrin receptor is in a range from 10⁻¹¹M to 10⁻⁶ M.

20. The humanized antibody of any one of embodiments 1-19, wherein theantibody does not specifically bind to the transferrin binding site ofthe transferrin receptor and/or wherein the antibody does not inhibitbinding of transferrin to the transferrin receptor.21. The humanized antibody of any one of embodiments 1-20, wherein theantibody is cross-reactive with extracellular epitopes of two or more ofa human, non-human primate and rodent transferrin receptor.22. A nucleic acid encoding the antibody in any one of embodiments 1-21.23. A vector comprising the nucleic acid of embodiment 22.24. A cell comprising the vector of embodiment 23.25. A method producing an anti-TfR antibody, comprising culturing thecell of embodiment 24 under conditions suitable for the expression ofthe antibody.26. A complex comprising the antibody of any one of embodiments 1-21covalently linked to a molecular payload.27. The complex of embodiment 26, wherein the molecular payload is adiagnostic agent or a therapeutic agent.28. The complex of embodiment 26, wherein the molecular payload is anoligonucleotide, a polypeptide, or a small molecule.29. The complex of any one of embodiments 26-28, wherein the antibodyand the molecular payload are linked via a linker.30. The complex of embodiment 29, wherein the linker is a cleavablelinker.31. The complex of embodiment 30, wherein the linker is comprises avaline-citrulline sequence.32. A composition comprising the antibody of any one of embodiments1-21, the nucleic acid of embodiment 22, the vector of embodiment 23, orthe complex of any one of embodiments 26-31.33. The composition of embodiment 32, further comprising apharmaceutically acceptable carrier.34. A method of detecting a transferrin receptor in a biological sample,comprising contacting the antibody of any one of embodiments 1-21 withthe biological sample and measuring binding of the antibody to thebiological sample.35. The method of embodiment 34, wherein the antibody is covalentlylinked to a diagnostic agent.36. The method of embodiment 35, wherein the biological sample isobtained from a human subject suspected of having or at risk for adisease associated with transferrin receptor.37. The method of embodiment 36, wherein the contacting step isperformed by administering the subject an effective amount of theanti-TfR antibody.38. A method of delivering a molecular payload to a cell, comprisingcontacting the complex of any one of embodiments 26-31 with the cell.39. The method of embodiment 38, wherein the cell is a muscle cell.40. The method of embodiment 38 or embodiment 39, wherein the cell is invitro.41. The method of embodiment 40, wherein the cell is in a subject.42. The method of embodiment 41, wherein the subject is human.43. A method of delivering a molecular payload to the brain or themuscle of a subject, comprising administering to the subject aneffective amount of the complex of any one of embodiments 26-31.44. The method of embodiment 43, wherein the administration isintravenous.45. A method of treating a disease, comprising administering to asubject an effective amount of the complex of any one of embodiments26-31, wherein the molecular payload is a therapeutic agent.46. The method of embodiment 45, wherein the disease is a neurologicaldisease and the molecular payload is a drug for treating a neurologicaldisease.47. The method of embodiment 45, wherein the disease is a muscle diseaseand the molecular payload is a drug for treating a muscle disease.48. The method of embodiment 47, wherein the muscle disease is a raremuscle disease or muscle atrophy.49. An antibody that binds to human transferrin receptor (TfR), whereinthe antibody comprises:

(i) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 76; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 75;

(ii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 69; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 71; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(iv) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 72; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 70;

(v) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 73; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 74;

(vi) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 73; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 75;

(vii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 76; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 74;

(viii) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 77; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 78;

(ix) a heavy chain variable region (VH) comprising an amino acidsequence at least 85% identical to SEQ ID NO: 79; and/or a light chainvariable region (VL) comprising an amino acid sequence at least 85%identical to SEQ ID NO: 80; or

(x) a heavy chain variable region (VH) comprising an amino acid sequenceat least 85% identical to SEQ ID NO: 77; and/or a light chain variableregion (VL) comprising an amino acid sequence at least 85% identical toSEQ ID NO: 80.

50. An antibody that binds to human transferrin receptor (TfR), whereinthe antibody has undergone pyroglutamate formation resulting from apost-translational modification.

EQUIVALENTS AND TERMINOLOGY

The disclosure illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the disclosure. Thus, it should be understood that although thepresent disclosure has been specifically disclosed by preferredembodiments, optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure.

In addition, where features or aspects of the disclosure are describedin terms of Markush groups or other grouping of alternatives, thoseskilled in the art will recognize that the disclosure is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group or other group.

It should be appreciated that, in some embodiments, sequences presentedin the sequence listing may be referred to in describing the structureof an oligonucleotide or other nucleic acid. In such embodiments, theactual oligonucleotide or other nucleic acid may have one or morealternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide ora DNA counterpart of an RNA nucleotide) and/or (e.g., and) one or moremodified nucleotides and/or (e.g., and) one or more modifiedinternucleotide linkages and/or (e.g., and) one or more othermodification compared with the specified sequence while retainingessentially same or similar complementary properties as the specifiedsequence.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein. Variations of thoseembodiments may become apparent to those of ordinary skill in the artupon reading the foregoing description.

The inventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A composition comprising a plurality ofanti-transferrin receptor (TfR) antibodies, wherein each anti-TfRantibody is a Fab comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 101 and a light chain comprising the amino acidsequence of SEQ ID NO:
 90. 2. The composition of claim 1, wherein theheavy chain of anti-TfR antibodies of the composition comprises anN-terminal pyroglutamate.
 3. The composition of claim 1, wherein thecomposition is an aqueous solution.
 4. The composition of claim 1,wherein the composition is lyophilized.
 5. The composition of claim 1,wherein the composition is present in a container.
 6. The composition ofclaim 1, wherein the composition is present in a column.
 7. Thecomposition of claim 1, wherein the composition is present in a subject.8. The composition of claim 1, wherein the composition is present in acell.
 9. The composition of claim 1, wherein the antibodies are bound toa solid support.